Enone reductases

ABSTRACT

The disclosure relates to engineered enone reductase polypeptides having improved properties, polynucleotides encoding the engineered polypeptides, related vectors, host cells, and methods for making the engineered enone reductase polypeptides. The disclosure also provides methods of using the engineered enone reductase polypeptides for chemical transformations.

The present application is a Divisional of U.S. patent application Ser.No. 14/504,558, filed Oct. 2, 2014, which is a Divisional of U.S. patentapplication Ser. No. 13/658,582, filed Oct. 23, 2012, which is aDivisional of U.S. patent application Ser. No. 12/646,907, filed Dec.23, 2009, which claims priority to U.S. Pat. Ser. No. 61/140,879, filedDec. 25, 2008, pursuant 35 U.S.C. §119(e), each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to polypeptides, polynucleotides encodingthe polypeptides, and methods of using the polypeptides andpolynucleotides.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith under 37 C.F.R.§1.821 in a computer readable form (CRF) via EFS-Web as file name“2-026US1_Sequence_Listing.txt” is herein incorporated by reference. Theelectronic copy of the Sequence Listing was created on Dec. 22, 2009with a file size of 542,201 bytes.

BACKGROUND

Enone reductase enzymes of the Old Yellow Enzyme (OYE) family catalyze arange of reductions of α,β unsaturated ketones, aldehydes, esters, andnitriles that are of potential industrial importance. One reaction ofinterest is the hydrogenation of nitroalkenes, which is present incertain industrial explosives and serves as intermediates for thesynthesis of a range of compounds, such as alkaloids, antibiotics, andbiocides. Accumulation of the nitronate can be enhanced by a Y196Fmutation of OYE (Meah and Massey, 2000, Proc Natl Acad Sci USA97(20):10733-8; Meah et al., 2001, Proc Natl Acad Sci USA98(15):8560-5), providing a attractive biocatalytic based production ofa nitronate and a useful alternative to the more complex chemicaltransformation needed to provide the same products.

Another useful reaction carried out by enone reductases is the reductionof 3,5,5-trimethyl-2-cyclohexene-1,4-dione to produce(6R)-2,2,6-trimethylcyclohexane-1,4-dione, also known as levodione,which is a useful chiral building block for synthesis of naturallyoccurring optically active carotenoid compounds, such as xanthoxin andzeaxanthin. Old Yellow Enzymes OYE1, OYE2 and OYE3 from yeastSaccharomyces pastorianus and Saccharomyces cerevisiae can also catalyzestereoselective reduction of α,β-unsaturated carbonyls, esters andnitriles. However, these enzymes can have a narrow substrate recognitionprofile and/or have stability properties that are not suited forcommercial applications. Thus, it is desirable to identify enonereductases having properties that may be advantageous, such as withrespect to substrate recognition profile, stereoselectivity, andstability.

SUMMARY OF THE INVENTION

The present disclosure relates to engineered enone reductasepolypeptides having altered enzyme properties relative to wildtype enonereductases. These engineered enone reductase polypeptides are capable ofreducing an α,β unsaturated compound, such as an α,β unsaturated ketone,aldehyde, ester or nitrile to the corresponding saturated ketone,aldehyde, ester or nitrile. In one aspect, the enone reductases of thedisclosure comprise a chimeric polypeptide of enone reductase 2 (ERED 2)of SEQ ID NO:4 and enone reductase 3 (ERED 3) of SEQ ID NO:6. In someembodiments, the chimeric polypeptide can also comprise a chimericpolypeptide of enone reductase 1 (ERED 1) of SEQ ID NO:2, enonereductase 2 (ERED 2) of SEQ ID NO:4, and enone reductase 3 (ERED 3) ofSEQ ID NO:6. The chimeric polypeptides described herein arecharacterized by improvements in thermal and/or solvent stability. Insome embodiments, the chimeric enone reductase polypeptides are stableto treatment conditions of 50% isopropanol at 30° C. and/or 10-20%isopropanol at 40° C. In some embodiments, the chimeric enone reductasesare capable of reducing or converting 1-cyclohex-2-enone tocyclohexanone. In some embodiments, the chimeric polypeptides are alsocapable of reducing or converting methyl (E)-but-2-enoate to methylbutanoate.

The present disclosure provides an enone reductase polypeptidecomprising an amino acid sequence that is selected from the groupconsisting of: (a) an amino acid sequence that is at least 80% identicalto a reference sequence selected from the group consisting of SEQ IDNOs: 8, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216 and 217; (b) an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions to a referencenucleic acid sequence selected from the group consisting of SEQ ID NOs:7, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, and215, or a complementary sequence thereof.

In some embodiments, the enone reductases of the present invention canhave one or more residue differences as compared to a reference enonereductase, such as the chimeric enone reductases characterized byimproved thermal and/or solvent stability. In some embodiments, theresidue differences can occur at one or more of the following residuepositions: X5; X10; X28; X38; X40; X44; X75; X83; X117; X119; X122;X124; X147; X148; X153; X154; X179; X209; X240; X248; X251; X252; X255;X259; X294; X295; X296; X297; X302; X304; X305; X311; X315; X330; X333;X339; X358; X369; X376; X379; X384; X386; X397; X399; and X400. Thepresence of certain amino acids at these residue positions areassociated with altered enzyme properties, including, among others,substrate recognition profile, stereoselectivity, and enzyme activity.Various amino acids that can occupy the specified positions and theassociated changes to enzyme properties are described in the detaileddescription.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (5-carvone) to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one. In some embodiments,the reductase polypeptide capable of converting(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 10, 14, 16,18, 46, 44, 68, 70, 72, 74, 76, 82, 86, 88, 90, 98, 102, 104, 108, 110,112, 114, 116, 118, 122, 126, 128, 130, 162 or 172.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess. In some embodiments, the engineered enone reductase capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess comprises an amino acid sequence that corresponds to the sequenceof SEQ ID NO: 20, 24, 28, 30, 32, 38, 48, 50, 52, 54, 56, 60, 64, 66,92, 144, 148, 152, 154, 156, or 158.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (R-carvone) to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess.

In some embodiments, the engineered enone reductase capable ofconverting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess comprises an amino acid sequence that correspondsto the sequence of SEQ ID NO: 10, 12, 14, 16, 18, 22, 26, 32, 34, 36,40, 42, 44, 46, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 94, 96, 98,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 134,136, 138, 140, 142, 146, 162, 166, 168, 170, 172, 174 or 186.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess.

In some embodiments, the enone reductase capable of converting(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess comprises an amino acid that corresponds to the sequence of SEQID NO: 28, 40, 144 or 148.

In some embodiments, the enone reductase is capable of reducing(Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate with at least 5 times the conversion rate ofSEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the enone reductase capable of reducing (Z)-ethyl2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoatecomprises an amino acid sequence that corresponds to the sequence of SEQID NO: 20, 22, 24, 26, 28, 30, 36, 40, 44, 48, 50, 54, 56, 82, 92, 94,96, 100, 128, 144, 146, 148, 152, 154, 156, 158, 170, 174, 176, 178,180, 182, or 184.

In some embodiments, the engineered enone reductase is capable ofreducing the α,β unsaturated ketone of8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 2 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the engineered enone reductase capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 2 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an amino acidsequence that corresponds to the sequence of SEQ ID NO: 12, 42, 68, 72,82, 86, 88, 98, 104, 106, 114, 118, 120, 122, 124, 126, 132, 136, 138,160, 162, 164, 166, 168, 170, 172, 176, 178, 180, 184, or 182.

In some embodiments, the engineered enone reductase is capable ofreducing 3-methylcyclohex-2-enone to 3-methylcyclohexanone with at least1 or 2 times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the enone reductase capable of reducing3-methylcyclohex-2-enone to 3-methylcyclohexanone with at least 1 timesthe conversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 18, 46,132, 174, 186 or 140.

In some embodiments, the enone reductase is capable of reducing2-methylcyclopente-2-none to 2-methylcyclopentanone.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered enone reductases described herein, andpolynucleotides that hybridize to such polynucleotides under highlystringent conditions. The polynucleotide can include promoters and otherregulatory elements useful for expression of the encoded engineeredenone reductases, and can utilize codons optimized for specific desiredexpression systems. Exemplary polynucleotides include, but are notlimited to, the sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, or 215.

In another aspect, the present disclosure provides host cells comprisingthe polynucleotides and/or expression vectors described herein, andmethods for culturing or incubating the host cells to produce theengineered enone reductase polypeptides. The host cells may be E. colior they may be a different organism. The host cells can be used for theexpression and isolation of the engineered enone reductase enzymesdescribed herein, or, alternatively, they can be used directly for theconversion of an α,β unsaturated substrate to the correspondingsaturated product.

In a further aspect, the present disclosure provides methods of usingthe enone reductase polypeptides described herein to reduce or convertan α,β unsaturated compound selected from the group consisting of aketone, a nitrile, an ester, and a nitrile to the correspondingsaturated ketone, nitrile, ester, or nitrile.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a reaction carried out by enone reductases of thepresent invention.

FIGS. 2A, 2B, 2C, 2D, and 2E provide an alignment of the nucleic acidsequence encoding ERED 1 (top row: SEQ ID NO:1), ERED 2 (middle row: SEQID NO:3), and ERED 3 (bottom row: SEQ ID NO:5), showing regions ofnucleic acid sequence homology. The underlined nucleotides representregions of differences in the sequences.

DETAILED DESCRIPTION

The present disclosure provides engineered enone reductase polypeptides,polynucleotides encoding the polypeptides and methods of using thepolypeptides for the reduction of α,β unsaturated ketones, aldehydes,esters, and nitrile compounds. For the descriptions herein and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a polypeptide” includes more than onepolypeptide, and reference to “a compound” refers to more than onecompound.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

The foregoing general description, including the drawings, and thefollowing detailed description are exemplary and explanatory only andare not restrictive of this disclosure.

ABBREVIATIONS

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter abbreviation One-letter abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When peptide sequences are presented as a stringof one-letter or three-letter abbreviations (or mixtures thereof), thesequences are presented in the N→C direction in accordance with commonconvention.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art, unless specifically defined herein. Accordingly, the followingterms are intended to have the following meanings.

“Enone reductase” and “ERED” are used interchangeably herein to refer toa polypeptide having a capability of reducing an α,β unsaturatedcompound to the corresponding saturated compound. More specifically,enone reductases are capable of reducing α,β unsaturated ketones,aldehydes, nitriles and esters. Enone reductases typically utilize acofactor reduced nicotinamide adenine dinucleotide (NADH) or reducednicotinamide adenine dinucleotide phosphate (NADPH) as the reducingagent. Enone reductases as used herein include naturally occurring (wildtype) enone reductases as well as non-naturally occurring engineeredpolypeptides generated by human manipulation.

“Chimeric” in the context of a gene or polypeptide refers to any gene orDNA or polypeptide that contains (1) DNA or polypeptide sequences thatare not found together in nature, or (2) sequences encoding parts ofproteins or proteins not naturally adjoined. Accordingly, a chimericgene or polypeptide may include sequences that are present in differentsources or from the same source rearranged in a manner not found innature. In some embodiments, the chimeric polypeptides comprise fusionproteins.

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” when used with reference to, e.g., a cell,nucleic acid, or polypeptide, refers to a material, or a materialcorresponding to the natural or native form of the material, that hasbeen modified in a manner that would not otherwise exist in nature, oris identical thereto but produced or derived from synthetic materialsand/or by manipulation using recombinant techniques. Non-limitingexamples include, among others, recombinant cells expressing genes thatare not found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, by the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WisconsinSoftware Package), or by visual inspection (see generally, CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)), all of whichreferences are incorporated herein by reference. Examples of algorithmsthat are suitable for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul etal., 1977, Nucleic Acids Res. 3389-3402, respectively, both of whichreferences are incorporated herein by reference. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915, which isincorporated herein by reference). Exemplary determination of sequencealignment and % sequence identity can employ the BESTFIT or GAP programsin the GCG Wisconsin Software package (Accelrys, Madison Wis.), usingdefault parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity.

In some embodiments, a “reference sequence” can be based on a primaryamino acid sequence, where the reference sequence is a sequence that canhave one or more changes in the primary sequence. For instance, a“reference sequence based on SEQ ID NO:8 having at the residuecorresponding to X117 an isoleucine” refers to a reference sequence inwhich the corresponding residue at X117 in SEQ ID NO:4 has been changedto an isoleucine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredenone reductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diasteromers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to an enone reductase polypeptide thatis capable of converting or reducing the substrate to the correspondingproduct with at least about 85% stereomeric excess.

“Stereospecificity” refers to the preferential conversion in a chemicalor enzymatic reaction of one stereoisomer over another.Stereospecificity can be partial, where the conversion of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is converted.

“Chemoselectivity” refers to the preferential formation in a chemical orenzymatic reaction of one product over another.

“Improved enzyme property” refers to a enone reductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference enone reductase. For the engineered enone reductasepolypeptides described herein, the comparison is generally made to thewild-type enone reductase enzyme, although in some embodiments, thereference enone reductase can be another improved engineered enonereductase. Enzyme properties for which improvement is desirable include,but are not limited to, enzymatic activity (which can be expressed interms of percent conversion of the substrate), thermal stability,solvent stability, pH activity profile, cofactor requirements,refractoriness to inhibitors (e.g., product inhibition),stereospecificity, and stereoselectivity (including enantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered enone reductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of ERED) ascompared to the reference enone reductase enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.5 times the enzymatic activity of the correspondingwild-type enone reductase enzyme, to as much as 2 times. 5 times, 10times, 20 times, 25 times, 50 times, 75 times, 100 times, or moreenzymatic activity than the naturally occurring enone reductase oranother engineered enone reductase from which the enone reductasepolypeptides were derived. In specific embodiments, the engineered enonereductase enzyme exhibits improved enzymatic activity in the range of1.5 to 50 times, 1.5 to 100 times greater than that of the parentreductase enzyme. It is understood by the skilled artisan that theactivity of any enzyme is diffusion limited such that the catalyticturnover rate cannot exceed the diffusion rate of the substrate,including any required cofactors. The theoretical maximum of thediffusion limit, or k_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹s⁻¹). Hence, any improvements in the enzyme activity of the enonereductase will have an upper limit related to the diffusion rate of thesubstrates acted on by the enone reductase enzyme. Enone reductaseactivity can be measured by any one of standard assays used formeasuring enone reductases, such as a decrease in absorbance orfluorescence of NADPH due to its oxidation with the concomitantreduction of the unsaturated bond, or by product produced in a coupledassay. Comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic reduction of the substrate to thecorresponding product. “Percent conversion” refers to the percent of thesubstrate that is reduced to the product within a period of time underspecified conditions. Thus, the “enzymatic activity” or “activity” of aenone reductase polypeptide can be expressed as “percent conversion” ofthe substrate to the product.

“Thermostable” refers to a enone reductase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

“Solvent stable” refers to a enone reductase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent, (e.g., isopropyl alcohol,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a enone reductase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to high orlow pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs)compared to the untreated enzyme.

“Thermo- and solvent stable” refers to a enone reductase polypeptidethat are both thermostable and solvent stable.

“Derived from” as used herein in the context of engineered enonereductase enzymes, identifies the originating enone reductase enzyme,and/or the gene encoding such enone reductase enzyme, upon which theengineering was based. For example, the engineered enone reductaseenzyme of SEQ ID NO: 8 was obtained by artificially recombining thegenes encoding enone reductase 1 (SEQ ID NO:2), enone reductase 2 (SEQID NO:4), and enone reductase 3 (SEQ ID NO:6). Thus, this engineeredenone reductase enzyme is “derived from” the wild-type polypeptides ofSEQ ID NO: 2, 4, and 6.

“Hydrophilic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic amino acid or residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-Pro (P) and L-His (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar amino acid or residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic amino acid or residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine” or L-Cys (C) is unusual in that it can form disulfide bridgeswith other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small amino acid or residue” refers to an amino acid or residue havinga side chain that is composed of a total of three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing amino acid or residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. The table below shows exemplary conservative substitutions.

TABLE 1 Residue Possible Conservative Mutations A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) P, H Other constrained (P, H) N, Q, S, T Other polar Y, W, FOther aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids making up the polypeptide while retaining enzymaticactivity and/or retaining the improved properties of an engineered enonereductase enzyme. Deletions can be directed to the internal portionsand/or terminal portions of the polypeptide. In various embodiments, thedeletion can comprise a continuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the improved engineered enone reductase enzymes comprise insertions ofone or more amino acids to the naturally occurring enone reductasepolypeptide as well as insertions of one or more amino acids to otherimproved enone reductase polypeptides. Insertions can be in the internalportions of the polypeptide, or to the carboxy or amino terminusInsertions as used herein include fusion proteins as is known in theart. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length enone reductasepolypeptide, for example the polypeptide of SEQ ID NO:8.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved enone reductase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved enone reductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure enone reductase compositionwill comprise about 60% or more, about 70% or more, about 80% or more,about 90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved enone reductases polypeptide is a substantially purepolypeptide composition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered enone reductase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theenone reductases enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariat analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the polynucleotide of interest. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide of interest, such as acoding sequence. The control sequence may comprise an appropriatepromoter sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of a polynucleotide ofinterest. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Cofactor regeneration system” refers to a set of reactants thatparticipate in a reaction that reduces the oxidized form of the cofactor(e.g., NADP⁺ to NADPH). Cofactors oxidized by the enonereductase-catalyzed reduction of the α,β unsaturated substrate areregenerated in reduced form by the cofactor regeneration system.Cofactor regeneration systems comprise a stoichiometric reductant thatis a source of reducing hydrogen equivalents and is capable of reducingthe oxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant.

“Optionally substituted” refers to the replacement of hydrogen with amonovalent or divalent radical. Suitable substitution groups include,for example, hydroxyl, nitro, amino, imino, cyano, halo, thio,thioamido, aminidino, imidino, oxo, oxamidino, methoxamidino, imidino,guanidine, sulfonamide, carboxyl, formyl, lower alkyl, lower alkoxy, andthe like.

“Lower alkyl” refers to branched or straight chain alkyl groupscomprising one to ten carbon atoms that are unsubstituted orsubstituted, e.g., with one or more halogen, hydroxyl, and the like.

“Lower alkoxy” refers to RO—, wherein R is lower alkyl. Representativeexamples of lower alkoxy groups include methoxy, ethoxy, t-butoxy,trifluoromethoxy, and the like.

“Aryl” refers to monocyclic and polycyclic aromatic groups and includeboth carbocyclic aryl groups and heterocylic aryl groups. Arylsubstituents of the present invention typically have from 3 to 14backbone carbon or hetero atoms. “Aralkyl” refers to an aryl moietysubstituted with an alkyl substituent.

Engineered Enone Reductase Polypeptides

The present disclosure provides engineered (or recombinant) enonereductase (ERED) polypeptides useful for reducing an α,β unsaturatedsubstrate compound to the corresponding saturated product compound. Morespecifically, the engineered enone reductase polypeptides are capable ofreducing α,β unsaturated ketones, aldehydes, nitriles and esters, asfurther discussed below. The engineered enone reductase polypeptidesdescribed herein may also be isolated. In the embodiments herein, theengineered EREDs have an improved property as compared to the naturallyoccurring enone reductase I enzyme (“ERED 1”) obtained fromSaccharomyces pastorianus (SEQ ID NO: 2); the enone reductase 2 (“ERED2”) obtained from Saccharomyces cerevisiae (SEQ ID NO:4); or the enonereductase 3 (“ERED 3”) obtained from Saccharomyces cerevisiae (SEQ IDNO:6), or improved as compared to another engineered enone reductase,such as the enone reductase of SEQ ID NO:8. The polynucleotide and/oramino acid sequence of the naturally occurring enone reductases aredescribed in the art as follows: ERED 1, also referred to as Old YellowEnzyme 1 (OYE1), is available as Genbank Accession No. Q02899.3GI:417431, and presented herein as SEQ ID NO:1 (polynucleotide) and SEQID NO:2 (amino acid); ERED 2, also referred to as Old Yellow Enzyme 2(OYE2), is available as Genbank Accession No. NP_012049.1 GI:6321973,presented herein as SEQ ID NO:3 (polynucleotide) and SEQ ID NO:4 (aminoacid); and ERED 3, also referred to a Old Yellow Enzyme 3 (OYE3), isavailable as Genbank Accession No. NP_015154.1 GI:6325086, and ispresented herein as SEQ ID NO:5 (polynucleotide) and SEQ ID NO:6 (aminoacid). The improved property includes improvements in one or more of thefollowing properties: enzyme activity, stability (e.g., solvent and/orthermo stability), stereoselectivity, stereospecificity, inhibitorresistance, and/or substrate recognition. In some embodiments, theengineered enone reductase polypeptides can have more than one improvedproperty, such as increased stability and substrate recognition.

In the characterizations of the enone reductase polypeptides herein, thepolypeptide can be described in reference to a sequence of a naturallyoccurring enone reductase (e.g., ERED 1, ERED 2, or ERED 3) or anotherengineered enone reductase, such as the engineered chimeric enonereductase of SEQ ID NO:8, or another invention enone reductasepolypeptide described herein. In some embodiments, the amino acidposition is determined in the reference polypeptide beginning from theinitiating methionine (M) residue, although it will be understood by theskilled artisan that this initiating methionine residue may be removedby biological processing machinery, such as in a host cell or in vitrotranslation system, to generate the mature protein lacking theinitiating methionine. The amino acid residue position at which aparticular amino acid is present or an amino acid change occurs in anamino acid sequence is sometimes described herein as “Xn” or “positionn”, where n refers to the residue position. A substitution mutation,where described, may be denoted by the symbol “→” or by conventionalnotation used by those skilled in the art, for example “W117V, where Wis the amino acid present in the reference sequence, the number 117 isthe residue position in the reference sequence, and V is the amino acidsubstitution.

The present disclosure provides engineered enone reductase polypeptidescomprising an amino acid sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the reference sequence selected from thegroup consisting of SEQ ID NOs: 8, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, and 217. Engineered enone reductasepolypeptides of the present invention do not include polypeptides havingthe amino acid sequences of SEQ ID NOS: 2, 4, and 6. Typically, theengineered enone reductase polypeptides have one or more differences ormodifications relative to the reference sequence as describedhereinbelow.

In some embodiments, the engineered enone reductases can have one ormore residue differences (i.e., modifications) as compared to areference sequence, such as the naturally occurring ERED 1, ERED 2, orERED 3, or an engineered ERED, such as the chimeric enzymes havingincreased thermo- and solvent stability. In some embodiments, themodifications are with respect to the sequence of SEQ ID NO: 8, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216. Insome embodiments, the modifications can be with respect to SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6, particularly with respect to SEQ ID NO:6. Insome embodiments, the enone polypeptides herein can have a number ofresidue differences as compared to the reference sequence (e.g.,naturally occurring polypeptide or an engineered polypeptide) to changethe properties of the enzyme, including, among others, enzymaticactivity, substrate recognition, inhibitor resistance,stereoselectivity, stereospecificity, and stability enhancements.

Residue differences or modifications include amino acid substitutions,deletions, and insertions. Any one or a combination of modifications canbe introduced into the naturally occurring or engineered polypeptide togenerate engineered polypeptides. In such embodiments, the number ofmodifications to the amino acid sequence can comprise one or more aminoacids, 2 or more amino acids, 3 or more amino acids, 4 or more aminoacids, 5 or more amino acids, 6 or more amino acids, 8 or more aminoacids, 10 or more amino acids, 15 or more amino acids, or 20 or moreamino acids, up to 10% of the total number of amino acids, up to 10% ofthe total number of amino acids, up to 15% of the total number of aminoacids, up to 20% of the total number of amino acids, or up to 30% of thetotal number of amino acids of the reference polypeptide sequence. Insome embodiments, the number of modifications to the naturally occurringpolypeptide or an engineered polypeptide that produces an improvedproperty may comprise from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9,1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30,1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 modifications of the referencesequence. In some embodiments, the number of modifications can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 amino acid residue positions. The modificationscan comprise insertions, deletions, substitutions, or combinationsthereof.

In some embodiments, the modifications comprise amino acid substitutionsto the reference sequence. Substitutions that can produce an improvedproperty may be at one or more amino acids, 2 or more amino acids, 3 ormore amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 8 or more amino acids, 10 or more amino acids, 15 ormore amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, up to 10% of the total number of amino acids, upto 20% of the total number of amino acids, or up to 30% of the totalnumber of amino acids of the reference enzyme sequence. In someembodiments, the number of substitutions to the naturally occurringpolypeptide or an engineered polypeptide that produces an altered enonereductase property can comprise from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acidsubstitutions of the reference sequence. In some embodiments, the numberof substitutions can be at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 amino acidresidue positions.

In some embodiments, the engineered enone reductase polypeptides of thedisclosure can have residue differences as compared to one of the areference sequences described herein at one or more of the followingresidue positions: X5; X10; X28; X38; X40; X44; X75; X83; X117; X119;X122; X124; X147; X148; X153; X154; X179; X209; X240; X248; X251; X252;X255; X259; X294; X295; X296; X297; X302; X304; X305; X311; X315; X330;X333; X339; X358; X369; X376; X379; X384; X386; X397; X399; and X400.The occupation by particular amino acid residues at these residuepositions is associated with changes to properties of the enonereductases and described in detail below as features of the enonereductase amino acid sequences.

In some embodiments, the residue differences at the specified residuepositions can be with respect to a reference chimeric enone reductasedescribed hereinbelow having the thermo- and/or solvent stability asdescribed herein, such as, for example, SEQ ID NO: 8, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216. That is,particular amino acid residues at the specified positions can be presentin an amino acid sequence encoding the chimeric enone reductasepolypeptides described hereinbelow, which exhibit thermo- and/or solventstability.

In some embodiments, the residue differences at the specified residuepositions can be with respect to a reference sequence of a naturallyoccurring enone reductase, such as the polypeptides of SEQ ID NO:2, 4,or 6. Thus, while the exemplary enone reductases presented herein arebased on a chimeric enone reductase, it is to be understood that variousresidue differences resulting in altered enzyme properties can beapplied to naturally occurring enone reductases of SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:6. Accordingly, in some embodiments, the engineeredenone reductases can also comprise an amino acid sequence that is atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to a reference sequence of SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6 having any one or more of the features orresidue differences described in detail herein.

In some embodiments, the choice of amino acid residues for the specifiedresidue positions can be based on the following features: residuecorresponding to X5 is a basic or acidic residue, particularly an acidicresidue; residue corresponding to X10 is a polar or constrained residue,particularly a polar residue; residue corresponding to X28 is analiphatic or constrained residue, particularly a constrained residue;residue corresponding to X38 is a polar or basic residue; residuecorresponding to X40 is a non-polar, aliphatic, aromatic, acidic orpolar residue, particularly an aliphatic, aromatic, acidic or polarresidue; residue corresponding to X44 is a constrained or aromaticresidue, particularly an aromatic residue; residue corresponding to X75is an aromatic, aliphatic or polar residue, particularly an aliphatic orpolar residue; residue corresponding to X83 is an aromatic, aliphatic,acidic, or basic residue, particularly an aliphatic, acidic, or basicresidue; residue corresponding to X117 is a cysteine or nonpolar,aliphatic, basic, acidic, polar, or aromatic residue; residuecorresponding to X119 is a constrained or aliphatic residue; residuecorresponding to X122 is an aliphatic or polar residue, particularly apolar residue; residue corresponding to X124 is an aromatic, non-polaror a constrained residue, particularly a non-polar or a constrainedresidue; residue corresponding to X147 is an acidic or non-polarresidue, particularly a non-polar residue; residue corresponding to X148is a polar or basic residue, particularly a basic residue; residuecorresponding to X153 is a basic or acidic residue, particularly a basicresidue; residue corresponding to X154 is a basic residue; residuecorresponding to X179 is a basic residue; residue corresponding to X209is a polar or acidic residue, particularly an acidic residue; residuecorresponding to X240 is a basic residue; residue corresponding to X248is an aromatic residue or cysteine, particularly cysteine; residuecorresponding to X251 is a cysteine, or aromatic, non-polar, aliphatic,acidic, basic, or polar residue; residue corresponding to X252 is apolar, aromatic or constrained residue, particularly an aromatic orconstrained residue; residue corresponding to X255 is a polar orconstrained residue, particularly a constrained residue; residuecorresponding to X259 is an acidic or non-polar residue, particularly anon-polar residue; residue corresponding to X294 is a polar or aliphaticresidue, particularly an aliphatic residue; residue corresponding toX295 is an acidic, polar, basic, or non-polar residue, particularlypolar, basic, or non-polar residue; residue corresponding to X296 is aconstrained, aromatic, non-polar, aliphatic, basic, acidic, or polarresidue, particularly an aromatic, non-polar, aliphatic, basic, acidic,or polar residue; residue corresponding to X297 is a polar, aromatic,non-polar, aliphatic, or basic residue, particularly aromatic,non-polar, aliphatic, or basic residue; residue corresponding to X302 isan acidic or non-polar residue, particularly a non-polar residue;residue corresponding to X304 is an acidic or basic residue,particularly a basic residue; residue corresponding to X305 is anaromatic or polar residue, particularly a polar residue; residuecorresponding to X311 is an acidic residue; residue corresponding toX315 is a constrained residue; residue corresponding to X330 is aconstrained, aromatic or basic residue, particularly an aromatic orbasic residue; residue corresponding to X333 is an aliphatic residue;residue corresponding to X339 is a basic or polar residue, particularlya polar residue; residue corresponding to X358 is an aliphatic residue;residue corresponding to X369 is a basic or acidic residue, particularlyan acidic residue; residue corresponding to X376 is an aromatic,non-polar, aliphatic, basic or acidic residue, particularly a non-polar,aliphatic, basic or acidic residue; residue corresponding to X379 is apolar or nonpolar residue, particularly a non-polar residue; residuecorresponding to X384 is a polar or aliphatic residue, particularly analiphatic residue; residue corresponding to X386 is an aromatic oracidic residue, particularly an acidic residue; residue corresponding toX397 is an aromatic or basic residue, particularly a basic residue;residue corresponding to X399 is a basic or acidic residue, particularlyan acidic residue; and residue corresponding to X400 is a polar residue.In some embodiments, the amino acid sequence can have at least two,three, four, five, six, seven, eight, nine, ten, or more of thefeatures. The sequence formula described herein as SEQ ID NO:217presents these features in the context of the chimeric enone reductaseof SEQ ID NO:8. In some embodiments, the enone reductase polypeptidescan have additionally one or more residue differences at residuepositions not specified by an X above as compared to a referencesequence, for example SEQ ID NO:8. In some embodiments, the differencescan be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50,1-55, or 1-60 residue differences at other amino acid residue positionsnot defined by X above. In some embodiments, the number of differencescan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22,24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences at other aminoacid residue positions. In some embodiments, the differences compriseconservative mutations.

In some embodiments, the amino acid residues at the specified positionscan be selected from one or more of the following features: residuecorresponding to X5 is E; residue corresponding to X10 is P; residuecorresponding to X28 is P; residue corresponding to X38 is N or S;residue corresponding to X40 is L, Y, E or S; residue corresponding toX44 is Y; residue corresponding to X75 is L or S; residue correspondingto X83 is L, R, V, I, K, E, or M; residue corresponding to X117 is C, L,A, M, V, I, N, Q, E, F, or S; residue corresponding to X119 is P or V;residue corresponding to X122 is T; residue corresponding to X124 is Gor P; residue corresponding to X147 is G; residue corresponding to X148is R; residue corresponding to X153 is E; residue corresponding to X154is R; residue corresponding to X179 is R; residue corresponding to X209is D; residue corresponding to X240 is R; residue corresponding to X248is C; residue corresponding to X251 is A; C, R, E, D, W, Y, R, S, V, G,L, or I; residue corresponding to X252 is H; residue corresponding toX255 is P; residue corresponding to X259 is G; residue corresponding toX294 is A; residue corresponding to X295 is T, N, or G; residuecorresponding to X296 is G, F, A, S, R, E, Q, K, or I; residuecorresponding to X297 is F; K; Y, W, G, A, or I; residue correspondingto X302 is G; residue corresponding to X304 is K; residue correspondingto X305 is S; residue corresponding to X311 is E; residue correspondingto X315 is P; residue corresponding to X330 is Y or R; residuecorresponding to X333 is A; residue corresponding to X339 is Q; residuecorresponding to X358 is A; residue corresponding to X369 is E; residuecorresponding to X376 is T, K, I, A, or E; residue corresponding to X379is G; residue corresponding to X384 is I; residue corresponding to X386is D; residue corresponding to X397 is R; residue corresponding to X399is E; and residue corresponding to X400 is T. In some embodiments, theenone reductase polypeptides can have additionally one or more residuedifferences at residue positions not specified by an X above as comparedto the reference sequence (e.g., SEQ ID NO:8). In some embodiments, thedifferences can be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other amino acidresidue positions not defined by X above. In some embodiments, thenumber of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residuedifferences at other amino acid residue positions. In some embodiments,the differences comprise conservative mutations.

In some embodiments, the engineered enone reductase amino acid sequencehas at least one or more of the following features: residuecorresponding to X40 is L, Y, E or S; residue corresponding to X83 is L,R, V, I, K, E, or M; residue corresponding to X117 is C; L; A; M; V; I;N; Q; E, F, or S; residue corresponding to X124 is G or P; residuecorresponding to X251 is A; C, R, E, D, W, Y, R, S, V, G, L, or I;residue corresponding to X296 is G, F, A, S, R, E, Q, K, or I; residuecorresponding to X297 is F; K; Y, W, G, A, or I; and residuecorresponding to X376 is T, K, I, A, or E.

In some embodiments, the engineered enone reductase amino acid sequencehas at least one of the following features: residue corresponding to X38is S and X117 is A; residue corresponding to X40 is L and X294 is A;residue corresponding to X75 is L and X297 is A; residue correspondingto X83 is I and X124 is P; residue corresponding to X83 is I and X251 isR; residue corresponding to X83 is I and X251 is A; residuecorresponding to X83 is I and X251 is V; residue corresponding to X83 isI and X251 is S; residue corresponding to X83 is K and X117 is I;residue corresponding to X83 is I and X117 is I; residue correspondingto X117 is A and X122 is T; residue corresponding to X117 is E and X386is D; residue corresponding to X148 is R and X251 is G; residuecorresponding to X248 is C and S297 is G; residue corresponding to X251is L and X379 is G; residue corresponding to X294 is A and X295 is G;residue corresponding to X296 is R and X330 is Y; residue correspondingto X296 is A and X297 is F; residue corresponding to X305 is S and X376is I; residue corresponding to X315 is P and X376 is T; residuecorresponding to X296 is K and X376 is A; residue corresponding to X10is P, X297 is G and X379 is G; residue corresponding to X38 is S, X83 isI and X117 is L; residue corresponding to X40 is S, X302 is G and X330is Y; residue corresponding to X83 is I, X251 is V and X295 is N;residue corresponding to X38 is S, X75 is L, X83 is I, X117 is I andX255 is P; residue corresponding to X38 is S, X83 is I, X117 is I andX119 is V; residue corresponding to X38 is S, X40 is Y, X83 is I andX117 is A; residue corresponding to X38 is S, X83 is I, X117 is A andX251 is D; residue corresponding to X38 is S, X83 is I, X117 is A andX251 is A; residue corresponding to X83 is I, X251 is A and X330 is Y;residue corresponding to X83 is I, X124 is G and X304 is K; residuecorresponding to X251 is E, X330 is R and X376 is I; residuecorresponding to X38 is S, X83 is I, X117 is I, X153 is E, X251 is W,X295 is T, X296 is F and X297 is Y; residue corresponding to X38 is S,X83 is I, X117 is I, X251 is A, X295 is N, X296 is F and X297 is W;residue corresponding to X38 is S, X83 is I, X117 is A, X251 is Y, X295is T, X296 is F and X297 is W; residue corresponding to X38 is S, X83 isI, X117 is I, X295 is T and X296 is A; residue corresponding to X5 is E,X44 is Y, X83 is I, X251 is A, X295 is N and X297 is G; residuecorresponding to X83 is I, X179 is R, X251 is C and X339 is Q; residuecorresponding to X38 is S, X83 is I, X117 is F and X251 is S; residuecorresponding to X38 is S, X83 is I, X117 is L, X209 is D, X251 is S,X376 is K and X400 is T; residue corresponding to X38 is S, X83 is I,X251 is S and X296 is G; residue corresponding to X38 is S, X83 is I,X117 is F, X251 is V and X376 is I; residue corresponding to X38 is S,X83 is I, X117 is F, X251 is S, X295 is N, X296 is G and X297 is F;residue corresponding to X38 is S, X83 is I, X251 is S, X295 is T, X296is S, X297 is F and X384 is I; residue corresponding to X28 is P, X83 isI, X117 is A and X251 is V; residue corresponding to X83 is I, X117 isN, X295 is T, X296 is G and X297 is F; residue corresponding to X83 isI, X117 is S, X251 is V, X296 is R and X297 is F; residue correspondingto X83 is I, X296 is S, X297 is F, X376 is I and X397 is R; residuecorresponding to X38 is S, X83 is I, X117 is N and X251 is V; residuecorresponding to X38 is S, X83 is I, X251 is S, X295 is T, X296 is G andX297 is F; residue corresponding to X38 is S, X83 is I, X154 is R, X251is V, X295 is T and X297 is F; residue corresponding to X38 is S, X83 isI, X117 is A and X330 is Y; residue corresponding to X38 is S, X83 is Iand X297 is Y; residue corresponding to X38 is S, X40 is E, X75 is S,X83 is I and X117 is I; residue corresponding to X38 is S, X83 is I andX117 is I; residue corresponding to X83 is E, X117 is I and X333 is A;residue corresponding to X251 is S, X296 is E, X297 is A and X311 is E;residue corresponding to X240 is R, X251 is S, X259 is G, X296 is Q andX297 is A; residue corresponding to X251 is I, X296 is S and X297 is F;residue corresponding to X251 is S, X297 is I and X358 is A; residuecorresponding to X251 is A, X296 is A; X297 is K and X399 is E; residuecorresponding to X251 is I, X296 is E, X297 is A and X376 is I; residuecorresponding to X296 is I; X297 is A, X333 is A and X376 is A; residuecorresponding to X296 is A, X297 is A; X330 is R and X376 is I; residuecorresponding to X147 is G, X296 is A, X297 is F, X330 is R and X376 isE; or residue corresponding to X297 is F, X369 is E and X376 is K. Insome embodiments, the enone reductase polypeptides can have additionallyone or more residue differences at other residue positions as comparedto the reference sequence (e.g., SEQ ID NO:8). In some embodiments, thedifferences can be 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other amino acidresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at other amino acidresidue positions. In some embodiments, the differences compriseconservative mutations.

In some embodiments, exemplary engineered enone reductase polypeptidescomprising an amino acid sequence with various features described hereincan be a sequence corresponding to SEQ ID NO: 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, or 188.

The present invention also provides an engineered enone reductasepolypeptide comprising an amino acid sequence encoded by a nucleic acidthat hybridizes under stringent hybridization conditions oversubstantially the entire length of a nucleic acid corresponding to areference polynucleotide sequence selected from the group consisting ofSEQ ID NOs: 7, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, and 215 and a complementary sequence thereof, where the aminoacid sequence is not SEQ ID NOS: 2, 4 or 6. The amino acid sequence ofthe encoded polypeptide may have one or more residue differencesrelative to the amino acid sequence encoded by the referencepolynucleotide sequences (i.e., SEQ ID NOs: 8, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, and 216, respectively) that aredescribed herein.

Typically, the encoded amino acid sequences comprise one or more of thefollowing features residue corresponding to X5 is E; residuecorresponding to X10 is P; residue corresponding to X28 is P; residuecorresponding to X38 is N or S; residue corresponding to X40 is L, Y, Eor S; residue corresponding to X44 is Y; residue corresponding to X75 isL or S; residue corresponding to X83 is L, R, V, I, K, E, or M; residuecorresponding to X117 is C, L, A, M, V, I, N, Q, E, F, or S; residuecorresponding to X119 is P or V; residue corresponding to X122 is T;residue corresponding to X124 is G or P; residue corresponding to X147is G; residue corresponding to X148 is R; residue corresponding to X153is E; residue corresponding to X154 is R; residue corresponding to X179is R; residue corresponding to X209 is D; residue corresponding to X240is R; residue corresponding to X248 is C; residue corresponding to X251is A; C, R, E, D, W, Y, R, S, V, G, L, or I; residue corresponding toX252 is H; residue corresponding to X255 is P; residue corresponding toX259 is G; residue corresponding to X294 is A; residue corresponding toX295 is T, N, or G; residue corresponding to X296 is G, F, A, S, R, E,Q, K, or I; residue corresponding to X297 is F; K; Y, W, G, A, or I;residue corresponding to X302 is G; residue corresponding to X304 is K;residue corresponding to X305 is S; residue corresponding to X311 is E;residue corresponding to X315 is P; residue corresponding to X330 is Yor R; residue corresponding to X333 is A; residue corresponding to X339is Q; residue corresponding to X358 is A; residue corresponding to X369is E; residue corresponding to X376 is T, K, I, A, or E; residuecorresponding to X379 is G; residue corresponding to X384 is I; residuecorresponding to X386 is D; residue corresponding to X397 is R; residuecorresponding to X399 is E; and residue corresponding to X400 is T.

In some embodiments, the enone reductases of the disclosure arecharacterized by increased stability as compared to the naturallyoccurring enone reductases ERED 1, ERED 2 or ERED 3. In someembodiments, the enone reductases are characterized by increasedthermostability and/or solvent stability, particularly with respect tothe ERED of SEQ ID NO:6. Thermostability can be readily assessed bysubjecting the polypeptide to a defined temperature under a set ofsolution conditions and measuring the enzyme activity remaining (i.e.,residual activity) following exposure to the defined temperature.Likewise, solvent stability can be readily assessed by subjecting thepolypeptide to a defined solvent under a defined condition and measuringthe enzyme activity remaining following exposure to the solvent. Thermalstability is advantageous where reaction with substrates are carried outat elevated temperatures and/or the enzymatic reaction carried out forlong time periods. Because certain substrates acted on by the engineeredenone reductases are prepared in organic solvents and the reactioncarried out in such solvents, solvent stability is also an advantageousenzyme property. Solvent stability can include stability in, amongothers, methanol (MEOH), isopropanol (IPA), tetrahydrofuran (THF),acetone (ACTN), acetonitrile (ACN), dimethylsulfoxide (DMSO),dimethylformamide (DMF), methyl tert butyl ether (MTBE), andn-butylacetate (nBuOAc). While the properties of thermal stability andsolvent stability can be separate, for example, an enone reductase thatis stable to elevated temperatures but not stable to isopropanol, insome embodiments, the enone reductases herein display both thermalstability and solvent stability.

In some embodiments, the engineered enone reductase polypeptides arecharacterized by increased stability under conditions of 50% isopropanolat 30° C. and/or 10-20% isopropanol at 40° C. as compared to thenaturally occurring enone reductase 3. In some embodiments, the enonereductases are characterized by increased stability in 50% isopropanolat 30° C. In some embodiments, the enone reductases are characterized byincreased stability in 10% isopropanol at 40° C. In some embodiments,the enone reductases are characterized by increased stability in 20%isopropanol at 40° C. The enone reductases are generally exposed to suchconditions for about 18 to 24 hrs to assess their stability under thespecified conditions.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:8.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:190.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:192.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:194.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:196.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:198.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:200.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:202.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:204.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:206.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:208.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:210.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:212.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:214.

In some embodiments, the engineered enone reductase with increasedstability comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:216.

In some embodiments, the enone reductases with increased thermal and/orsolvent stability can comprise a chimeric polypeptide of enone reductase2 (ERED 2) of SEQ ID NO:4, and enone reductase 3 (ERED 3) of SEQ IDNO:6. In some embodiments, the chimera can also include sequences fromenone reductase 1 (ERED 1) of SEQ ID NO:2 such that the polypeptide withincreased thermal and/or solvent stability comprises a fusionpolypeptide of amino acid sequences derived from ERED 1, ERED 2, andERED 3. These chimeric enzymes can be obtained based on in vitrorecombination at regions of nucleic acid sequence homology between thedifferent enone reductase genes. Techniques to obtain chimeric enzymesare described in the art, such as that disclosed in Riechmann andWinter, 2000, Proc Natl Acad Sci USA 97(18):10068-10073; Crameri et al.,1998, Nature (London) 391:288-291; Ostermeier et al., 1999, Proc NatlAcad Sci USA 96:3562-3567; and Altamirano et al., 2000, Nature (London)403:617-622; Stemmer, W. P. C., 1994, Nature 370:389-391; Stemmer, W. P.C., 1994, Proc Natl Acad Sci. USA 91:10751; U.S. Pat. No. 5,605,793;U.S. Pat. No. 5,811,238; and U.S. Pat. No. 5,830,721; all of whichreferences are incorporated herein by reference. The regions ofpolynucleotide sequence identity between ERED 1, ERED 2, and ERED 3 areshown in FIGS. 2A, 2B, 2C, 2D, and 2E. Once chimeras with the desiredproperties are obtained, general recombinant DNA methodology can be usedto generate additional chimeric polypeptides with varying recombinationpoints in the polypeptide sequence.

In some embodiments, the chimera can have the following basic structureS^(A)˜S^(B)wherein

S^(A) comprises a segment initiating from residues of about 1 andterminating at residues of about 120 to 150 and is derived from ERED 1,ERED 2, ERED 3, or combinations thereof; and

S^(B) comprises a segment initiating from residues of about 121 to 151and terminating at residue of about 400 and is derived from ERED 2, ERED3, or combinations thereof. The chimera with segments S^(A) and S^(B)can be further subdivided into sub-segments, where the sub-segments canbe derived from the specified enone reductases and combinations thereof.It is also to be understood that the chimeric structure does not excludea fusion polypeptide where a contiguous portion of a specific enonereductase beginning in segment S^(A) extends to segment S^(B), therebyforming a continuous sequence across the segments as found in thenaturally occurring enone reductase. The same applies to any of thechimeric structures described herein.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 14 to 20 and is derived from ERED 1;

S² comprises a segment initiating from residues of about 15 to 21 andterminating at residues of about 124 to 130 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 125 to 131 andterminating at residues of about 272 to 278 and is derived from ERED 2;and

S⁴ comprises a segment initiating from residues of about 273 to 279 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴˜S⁵˜S⁶wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 16 to 22 and is derived from ERED 1;

S² comprises a segment initiating from residues of about 17 to 23 andterminating at residues of about 56 to 62 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 57 to 63 andterminating at residues of about 106 to 112 and is derived from ERED 2;

S⁴ comprises a segment initiating from residues of about 107 to 113 andterminating at residues of about 138 to 144 and is derived from ERED 3;

S⁵ comprises a segment initiating from residues of about 139 to 145 andterminating at residues of about 296 to 302 and is derived from ERED 2;and

S⁶ comprises a segment initiating from residues of about 297 to 303 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 56 to 62 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 57 to 63 andterminating at residues of about 272 to 278 and is derived from ERED 2;and

S³ comprises a segment initiating from residues of about 273 to 279 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 106 to 112 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 107 to 113 andterminating at residue of about 247 to 253 and is derived from ERED 2;and

S³ comprises a segment initiating from residues of about 248 to 254 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 77 to 83 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 78 to 84 andterminating at residues of about 173 to 179 and is derived from ERED 2;

S³ comprises a segment initiating from residues of about 174 to 180 andterminating at residues of about 334-340 and is derived from ERED 3:

S⁴ comprises a segment initiating from residues of about 335 to 341 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 26 to 32 and is derived from ERED 2;

S² comprises a segment initiating from residues of about 27 to 33 andterminating at residues of about 100 to 106 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 101 to 107 andterminating at residues of about 120-126 and is derived from ERED 1; and

S⁴ comprises a segment initiating from residues of about 121 to 127 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴˜S⁵˜S⁶wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 16 to 22 and is derived from ERED 2;

S² comprises a segment initiating from residue of about 17 to 23 andterminating at residues of about 65 to 71 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 66 to 73 andterminating at about residues 100 to 106 and is derived from ERED 2:

S⁴ comprises a segment initiating from residues of about 101 to 107 andterminating at residues of about 138 to 144 and is derived from ERED 3;

S⁵ comprises a segment initiating from residues of about 139 to 145 andterminating at residues of about 173 to 179 and is derived from ERED 2;and

S⁶ comprises a segment initiating from residues of about 173 to 180 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 16 to 22 and is derived from ERED 2;

S² comprises a segment initiating from residues of about 17 to 23 andterminating at residues of about 247 to 253 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 248 to 254 andterminating at residues of about 296 to 302 and is derived from ERED 2;and

S⁴ comprises a segment initiating from residues of about 297 to 303 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴˜S⁵wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 65 to 71 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 66 to 72 andterminating at residues of about 173 to 179 and is derived from ERED 2;

S³ comprises a segment initiating from residues of about 174 to 180 andterminating at residues of about 237 to 243 and is derived from ERED 3:

S⁴ comprises a segment initiating from residues of about 238 to 244 andterminating at residues of about 294 to 300 and is derived from ERED 2;and

S⁵ comprises a segment initiating from residues of about 295 to 301 andterminating at residues of about 400 and is derived from ERED 3.

In some embodiments, the enone reductase comprises the structure:S¹˜S²wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 308 to 314 and is derived from ERED 3;and

S² comprises a segment initiating from residues of about 309 to 315 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴˜S⁵wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 40 to 46 and is derived from ERED 2;

S² comprises a segment initiating from residue of about 41 to 47 andterminating at residues of about 53 to 59 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 54 to 60 andterminating residues of about 206 to 212 and is derived from ERED 2:

S⁴ comprises a segment initiating from residues of about 207 to 213 andterminating at residues of about 257 to 263 and is derived from ERED 3;and

S⁵ comprises a segment initiating from residues of about 258 to 264 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 16 to 24 and is derived from ERED 2;

S² comprises a segment initiating from residues of about 17 to 25 andterminating at residues of about 124 to 130 and is derived from ERED 3;and

S³ comprises a segment initiating from residues of about 125 to 131 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 53 to 59 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 54 to 60 andterminating at residues of about 71 to 77 and is derived from ERED 1;

S³ comprises a segment initiating from residues of about 72 to 78 andterminating at residues of about 115 to 121 and is derived from ERED 3;and

S⁴ comprises a segment initiating from residues of about 116 to 120 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 40 to 46 and is derived from ERED 3;

S² comprises a segment initiating from residues of about 41 to 47 andterminating at residues of about 109 to 115 and is derived from ERED 1;and

S³ comprises a segment initiating from residues of about 110 to 116 andterminating at residue of about 400 and is derived from ERED 2.

In some embodiments, the enone reductase comprises the structure:S¹˜S²˜S³˜S⁴˜S⁵˜S⁶˜S⁷wherein

S¹ comprises a segment initiating from residue of about 1 andterminating at residues of about 16 to 22 and is derived from ERED 2;

S² comprises a segment initiating from residues of about 17 to 23 andterminating at residues of about 31 to 36 and is derived from ERED 3;

S³ comprises a segment initiating from residues of about 32 to 37 andterminating at residues of about 69 to 75 and is derived from ERED 1:

S⁴ comprises a segment initiating from residues of about 70 to 76 andterminating at residues of about 77 to 83 and is derived from ERED 2;

S⁵ comprises a segment initiating from residues of about 78 to 84 andterminating at residues of about 106 to 112 and is derived from ERED 3:

S⁶ comprises a segment initiating from residues of about 107 to 113 andterminating at about residues of about 173 to 179 and is derived fromERED 2; and

S⁷ comprises a segment initiating from residues of about 174 to 180 andterminating at residue of about 400 and is derived from ERED 3.

In some embodiments, the engineered enone reductase is characterized byincreased stability in 50% isopropanol at 30° C. as compared to thepolypeptide of SEQ ID NO:6 and comprises an amino acid sequencecorresponding to SEQ ID NO: 8, 192, 194, 200, 204, or 216.

In some embodiments, the engineered enone reductase is characterized byincreased stability in 10% isopropanol at 40° C. as compared to thepolypeptide of SEQ ID NO:6 and comprises an amino acid sequencecorresponding to SEQ ID NO: 8, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, or 216.

In some embodiments, the enone reductase is characterized by increasedstability in 20% isopropanol at 40° C. and comprises an amino acidsequence corresponding to SEQ ID NO: 8, 192, 194, 196, 198, 200, 202, or210.

In some embodiments, the enone reductase is characterized by increasedstability under conditions of 50% isopropanol at 30° C. and 10%isopropanol at 40° C. as compared to the polypeptide of SEQ ID NO:6. Insome embodiments, the enone reductase stable to elevated temperature andisopropanol comprises a sequence corresponding to SEQ ID NO: 8, 192,194, 200, 204, or 216.

In some embodiments, the engineered enone reductases are capable ofreducing an optionally substituted cyclohexenone to the correspondingcyclohexanone. For example, these embodiments include engineeredreductases that are capable of reducing 1-cyclohex-2-enone of formula(III) to the cyclohexanone of formula (IV):

The naturally occurring ERED 1 is capable of acting on the1-cyclohex-2-enone while the naturally occurring ERED 2 and ERED 3 showminimal activity towards this substrate. Given that the naturallyoccurring ERED 1 is not stable to temperature and organic solvents, noneof the naturally occurring enone reductases display significanttemperature and/or solvent stability and activity towards the substrateof formula (III). In some embodiments, the engineered enone reductasesdescribed herein are thermo and solvent stable, and have the capabilityof reducing 1-cyclohex-2-enone to the cyclohexanone. The latter is usedin the production of adipic acid for the synthesis of nylon andcaprolactam, as well serving as a solvent in various organic synthesisprocesses, such as the synthesis of polymers.

In some embodiments, the engineered enone reductases are also capable ofreducing an optionally alkenoate to the corresponding alkanoate. Forexample, these embodiments include engineered reductases that arecapable of reducing methyl (E)-but-2-enoate of formula (V) to methylbutanoate of formula (VI):

and have improved properties of thermal stability and/or solventstability as compared to the naturally occurring enone reductases of SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:6, particularly the polypeptide ofSEQ ID NO:6. Enone reductases capable of reducing both1-cyclohex-2-enone and methyl (E)-but-2-enoate have expanded substratespecificity and are good starting points for generation of otherengineered enone reductases with improved properties. As will beapparent to the skilled artisan, various chimeric enone reductasescharacterized by thermal and/or solvent stability, and capable ofconverting 1-cyclohex-2-enone to cyclohexanone and/or methyl(E)-but-2-enoate to methyl butanoate can be screened and obtained usingthe guidance provided in the present disclosure.

In some embodiments, the engineered enone reductase is capable ofreducing an optionally substituted cyclohexenone to the correspondingcyclohexanone. For example, these embodiments include engineered enonereductase polypeptides that are capable of converting the α,βunsaturated ketone of (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one(VII) to (2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (VIII) asfollows:

In some embodiments, the engineered enone reductase is capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one. In some embodiments,the engineered enone reductase is capable of converting(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one the with at least 1.5times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the engineered enone reductase capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one comprises an aminoacid sequence with at least one of the following features: residuecorresponding to X83 is V; residue corresponding to X83 is L; residuecorresponding to X83 is R; residue corresponding to X119 is P; residuecorresponding to X251 is C; residue corresponding to X251 is A; residuecorresponding to X251 is R; residue corresponding to X296 is G; residuecorresponding to X83 is I and X124 is P; residue corresponding to X40 isL and X294 is A; residue corresponding to X83 is I, X124 is G, X304 isK; residue corresponding to X296 is A and X297 is F; residuecorresponding to X251 is L and X379 is G; residue corresponding to X296is R and X330 is Y; residue corresponding to X294 is A and X295 is G;residue corresponding to X83 is I and X251 is R; residue correspondingto X83 is I and X251 is A; residue corresponding to X148 is R and X251is G; residue corresponding to X83 is I and X251 is V; residuecorresponding to X83 is I and X251 is S; residue corresponding to X83 isI, X251 is A and X330 is Y; residue corresponding to X83 is I, X251 is Vand X295 is N; residue corresponding to X83 is I, X179 is R, X251 is Cand X339 is Q; residue corresponding to X251 is A, X296 is A, X297 is Kand X399 is E; residue corresponding to X38 is S, X83 is I, X251 is Sand X296 is G; residue corresponding to X5 is E, X44 is Y, X83 is I,X251 is A, X295 is N and X297 is G; residue corresponding to X38 is S,X83 is I, X251 is S, X295 is T, X296 is G and X297 is F; residuecorresponding to X38 is S, X83 is I, X251 is S, X295 is T, X296 is S,X297 is F and X384 is I; residue corresponding to X38 is S, X83 is I,X117 is F, X251 is S, X295 is N, X296 is G and X297 is F; or residuecorresponding to X38 is S, X83 is I, X117 is N and X251 is V.

In some embodiments, the reductase polypeptide capable of converting(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 10, 14, 16,18, 46, 44, 68, 70, 72, 74, 76, 82, 86, 88, 90, 98, 102, 104, 108, 110,112, 114, 116, 118, 122, 126, 128, 130, 162 or 172.

In some embodiments, the enone reductase is capable of converting(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to the(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 2 timesthe conversion rate of SEQ ID NO:6 or SEQ ID NO:8. In some embodiments,the enone reductase comprises an amino acid sequence that corresponds tothe sequence of SEQ ID NO: 16, 44, 46, 68, 70, 72, 74, 76, 82, 90, 98,108, 112, 114, 116, 118, 130 or 172.

In some embodiments, the enone reductase is capable of converting the(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to the(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 2.5times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8. In someembodiments, the enone reductase comprises an amino acid sequence thatcorresponds to the sequence of SEQ ID NO: 68, 72, 76, 90, 98, 130 or172.

In some embodiments, the engineered enone reductase is capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess and 1.5 times the conversion rate of SEQ ID NO:6.In these embodiments, the enone reductase can comprise an amino acidsequence having at least one of the following features: residuecorresponding to X83 is L; residue corresponding to X83 is R; residuecorresponding to X119 is P; residue corresponding to X251 is C; residuecorresponding to X251 is A; residue corresponding to X251 is L and X379is G; residue corresponding to X83 is I and X124 is P; residuecorresponding to X83 is I and X251 is R; residue corresponding to X83 isI and X251 is A; residue corresponding to X148 is R and X251 is G;residue corresponding to X83 is I and X251 is V; residue correspondingto X83 is I and X251 is S; residue corresponding to X83 is I, X251 is Aand X330 is Y; residue corresponding to X83 is I, X251 is V and X295 isN; residue corresponding to X38 is S, X83 is I, X251 is S and X296 is G;residue corresponding to X251 is A, X296 is A, X297 is K and X399 is E;residue corresponding to X83 is I, X179 is R, X251 is C and X339 is Q;residue corresponding to X5 is E, X44 is Y, X83 is I, X251 is A, X295 isN and X297 is G; residue corresponding to X38 is S, X83 is I, X251 is S,X295 is T, X296 is G and X297 is F; or residue corresponding to X38 isS, X83 is I, X117 is F, X251 is S, X295 is N, X296 is G and X297 is F.

In some embodiments, the engineered enone reductase capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess and 1.5 times the conversion rate of SEQ ID NO:6comprises an amino acid sequence that corresponds to the sequence of SEQID NO: 16, 14, 46, 68, 70, 72, 74, 76, 82, 86, 90, 98, 104, 108, 110,112, 114, 116, 130 or 172.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (VII) to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (VIIIb) indiastereomeric excess, as illustrated in the following reaction:

In some embodiments, the engineered enone reductase capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess comprises an amino acid sequence with at least one of thefollowing features: residue corresponding to X117 is Q; residuecorresponding to X117 is A; residue corresponding to X117 is I; residuecorresponding to X117 is C; residue corresponding to X117 is V; residuecorresponding to X83 is I and X117 is I; residue corresponding to X117is A and X122 is T; residue corresponding to X38 is S and X117 is A;residue corresponding to X38 is S, X83 is I, and X117 is I; residuecorresponding to X38 is S, X83 is I, and X117 is L; residuecorresponding to X28 is P, X83 is I, X117 is A, and X251 is V; residuecorresponding to X38 is S, X83 is I, X117 is A and X251 is D; residuecorresponding to X38 is S, X83 is I, X117 is A and X251 is A; residuecorresponding to X38 is S, X40 is Y, X83 is I and X117 is A; residuecorresponding to X38 is S, X83 is I, X117 is A and X330 is Y; residuecorresponding to X38 is S, X83 is I, X117 is I and X119 is V; residuecorresponding to X38 is S, X40 is E, X75 is S, X83 is I and X117 is I;residue corresponding to X38 is S, X83 is I, X117 is I, D295 is T andX296 is A; residue corresponding to X38 is S, X75 is L, X83 is I, X117is I and X255 is P; or residue corresponding to X38 is S, X83 is I, X117is I, X251 is A, X295 is N, X296 is F and X297 is W.

In some embodiments, the engineered enone reductase capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess comprises an amino acid sequence that corresponds to the sequenceof SEQ ID NO: 20, 24, 28, 30, 32, 38, 48, 50, 52, 54, 56, 60, 64, 66,92, 144, 148, 152, 154, 156, or 158.

In some embodiments, the engineered enone reductase is capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 80%diastereomeric excess and comprises an amino acid sequence thatcorresponds to the sequence of SEQ ID NO: 20, 24, 28, 30, 48, 50, 52,54, 64, 144, 148, 152, 154, or 158.

In some embodiments, the engineered enone reductase is capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess and comprises an amino acid sequence thatcorresponds to the sequence of SEQ ID NO: 24, 28, 30, 48, 50, 54, 144,148, 152, or 154.

In some embodiments, the engineered enone reductase is capable ofconverting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with greater than 95%diastereomeric excess and comprises an amino acid sequence thatcorresponds to the sequence of SEQ ID NO: 24, 48, 50, 144, 148, 152, or154.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (R-carvone) (IX) to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (X) with at least 90%diastereomeric excess, as illustrated below:

In some embodiments, the enone reductase polypeptides capable ofconverting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one comprises an aminoacid sequence with at least one of the following features: residuecorresponding to X38 is N; residue corresponding to X83 is V; residuecorresponding to X83 is L; residue corresponding to X83 is R residuecorresponding to X117 is M; residue corresponding to X117 is N; residuecorresponding to X117 is L; residue corresponding to X117 is I; residuecorresponding to X251 is A; residue corresponding to X251 is C; residuecorresponding to X251 is R; residue corresponding to X252 is H; residuecorresponding to X296 is G; residue corresponding to X297 is K; residuecorresponding to X297 is Y; residue corresponding to X297 is F; residuecorresponding to X83 is I and X124 is P; residue corresponding to X83 isI and X251 is R; residue corresponding to X83 is I and X251 is S;residue corresponding to X305 is S and X376 is I; residue correspondingto X40 is L and X294 is A; residue corresponding to X315 is P and X376is T; residue corresponding to X75 is L and X297 is A; residuecorresponding to X296 is A and X297 is F; residue corresponding to X248is C and X297 is G; residue corresponding to X251 is L and X379 is G;residue corresponding to X296 is K and X376 is A; residue correspondingto X296 is R and X330 is Y; residue corresponding to X294 is A and X295is G; residue corresponding to X83 is I and X251 is A; residuecorresponding to X148 is R and X251 is G; residue corresponding to X83is I and X251 is V; residue corresponding to X117 is E and X386 is D;residue corresponding to X297 is F, X369 is E and X376 is K; residuecorresponding to X83 is I, X124 is G and X304 is K; residuecorresponding to X40 is S, X302 is G and X330 is Y; residuecorresponding to X251 is I, X296 is S and X297 is F; residuecorresponding to X38 is S, X83 is I and X297 is Y; residue correspondingto X251 is S, X297 is I and X358 is A; residue corresponding to X83 isI, X251 is A and X330 is Y; residue corresponding to X83 is I, X251 isV, and X295 is N; residue corresponding to X83 is I, X179 is R, X251 isC and X339 is Q; residue corresponding to X251 is A, X296 is A, X297 isK and X399 is E; residue corresponding to X5 is E, X44 is Y, X83 is I,X251 is A, X295 is N and X297 is G; residue corresponding to X38 is S,X83 is I, X154 is R, X251 is V, X295 is T and X297 is F; residuecorresponding to X38 is S, X83 is I, X251 is S, X295 is T, X296 is S,X297 is F and X384 is I; residue corresponding to X83 is I, X117 is S,X251 is V, X296 is R and X297 is F; residue corresponding to X38 is S,X83 is I, X251 is S and X296 is G; residue corresponding to X38 is S,X83 is I, X117 is F and X251 is S; residue corresponding to X38 is S,X83 is I, X251 is S, X295 is T, X296 is G and X297 is F; residuecorresponding to X38 is S, X83 is I, X117 is F, X251 is S, X295 is N,X296 is G and X297 is F; residue corresponding to X38 is S, X83 is I,X117 is L, X209 is D, X251 is S, X376 is K, and 400 is T; residuecorresponding to X38 is S, X83 is I, X117 is N and X251 is V; or residuecorresponding to X83 is I, X117 is N, X295 is T, X296 is G and X297 isF.

In some embodiments, the engineered enone reductase capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with at least 90%diastereomeric excess comprises an amino acid sequence that correspondsto the sequence of SEQ ID NO: 10, 12, 14, 16, 18, 22, 26, 32, 34, 36,40, 42, 44, 46, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 94, 96, 98,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 134,136, 138, 140, 142, 146, 162, 166, 168, 170, 172, 174 or 186.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with greater than 95%diastereomeric excess.

In some embodiments, the engineered enone reductase polypeptide capableof converting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one with greater than 95%diastereomeric excess comprises an amino acid sequence that correspondsto the sequence of SEQ ID NO: 34 or 186.

In some embodiments, the engineered enone reductase is capable ofconverting the α,β unsaturated ketone of(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (R-carvone) (IX) to(2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (Xb) in diastereomericexcess, as illustrated in the following reaction:

In some embodiments, the enone reductase polypeptides capable ofconverting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one comprises an aminoacid sequence with at least one of the following features: residuecorresponding to X117 is A and X122 is T; residue corresponding to X315is P and X376 is T; residue corresponding to X38 is S and X117 is A;residue corresponding to X38 is S, X83 is I, X117 is A and X330 is Y.

In some embodiments, the enone reductase polypeptides capable ofconverting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one to(2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one in diastereomericexcess comprises an amino acid that corresponds to the sequence of SEQID NO:28, 40, 144 or 148.

In some embodiments, the enone reductase polypeptide of the presentinvention is capable of reducing an optionally substituted arylalkenoneto an optionally substituted arylalkanone. For example, the presentinvention provides engineered enone reductase polypeptides that arecapable of reducing the arylalkenone of Formula XI to the arylalkanoneof Formula XII:

wherein for (XI) and (XII), R¹ and R² are each independently selectedfrom the group consisting of CN, C(O)R¹¹, C(O)OR¹¹, an alkyl (such as,for example, a lower alkyl), and H, wherein R¹¹ is selected from thegroup consisting of H and an alkyl (such as, for example, a loweralkyl), and wherein R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each independentlyselected from H, an alkyl (such as, for example, a lower alkyl), analkoxy (such as, for example, a lower alkoxy), a hydroxyl, and a halide.Typically, only one of R¹ and R² is CN and only one of R¹ and R² isC(O)R¹¹ or C(O)OR¹¹. Usually, at least one of R¹ and R² is C(O)R¹¹ orC(O)OR¹¹. In some embodiments, R² is an alkyl, such as, for example, alower alkyl (i.e., methyl, propyl, isopropyl, and the like).

In some embodiments, the engineered enone reductase is capable ofreducing the α,β unsaturated nitrile (Z)-ethyl2-cyano-3-phenylbut-2-enoate (XIII) to ethyl 2-cyano-3-phenylbutanoate(XIV) as follows:

In some embodiments, the enone reductase is capable of reducing(Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate with at least 5 times the conversion rate ofSEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the enone reductase capable of reducing (Z)-ethyl2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoatecomprises an amino acid sequence with at least one of the followingfeatures: residue corresponding to X117 is N; residue corresponding toX117 is L; residue corresponding to X117 is A; residue corresponding toX117 is M; residue corresponding to X117 is C; residue corresponding toX117 is V; residue corresponding to X296 is G; residue corresponding toX38 is S and X117 is A; residue corresponding to X83 is I and X117 is I;residue corresponding to X117 is A and X122 is T; residue correspondingto X305 is S and X376 is I; residue corresponding to X315 is P and X376is T; residue corresponding to X296 is K and X376 is A; residuecorresponding to X38 is S, X83 is I and X297 is Y; residue correspondingto X38 is S, X83 is I and X117 is I; residue corresponding to X83 is I,X124 is G and X304 is K; residue corresponding to X83 is E, X117 is Iand X333 is A; residue corresponding to X251 is E, X330 is R and X376 isI; residue corresponding to X38 is S, X83 is I, X117 is A and X251 is D;residue corresponding to X38 is S, X83 is I, X117 is A, X251 is A;residue corresponding to X296 is I, X297 is A, X333 is A and X376 is A;residue corresponding to X296 is A, X297 is A, X330 is R and X376 is I;residue corresponding to X83 is I, X296 is S, X297 is F, X376 is I andX397 is R; residue corresponding to X147 is G, X296 is A, X297 is F,X330 is R and X376 is E; residue corresponding to X251 is I, X296 is E,X297 is A, X376 is I; residue corresponding to X38 is S, X75 is L, X83is I, X117 is I and X255 is P; residue corresponding to X38 is S, X83 isI, X117 is I and X119 is V; residue corresponding to X83 is I, X117 isS, X251 is V, X296 is R, X297 is F; residue corresponding to X38 is S,X83 is I, X251 is S and X296 is G; residue corresponding to X38 is S,X83 is I, X117 is A, X330 is Y; residue corresponding to X38 is S, X40is E, X75 is S, X83 is I and X117 is I; residue corresponding to X83 isI, X117 is N, X295 is T, X296 is G and X297 is F; or residuecorresponding to X28 is P, X83 is I, X117 is A and X251 is V.

In some embodiments, the enone reductase capable of reducing (Z)-ethyl2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoatecomprises an amino acid sequence that corresponds to the sequence of SEQID NO: 20, 22, 24, 26, 28, 30, 36, 40, 44, 48, 50, 54, 56, 82, 92, 94,96, 100, 128, 144, 146, 148, 152, 154, 156, 158, 170, 174, 176, 178,180, 182, or 184.

In some embodiments, the enone reductase is capable of reducing(Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate with at least 10 times the conversion rate ofSEQ ID NO:6 or SEQ ID NO:8. In some embodiments, the enone reductasecapable of reducing the α,β unsaturated nitrile of (Z)-ethyl2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoate with atleast 10 times the conversion rate of SEQ ID NO:6 comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 20, 24, 28,30, 44, 48, 50, 54, 92, 94, 100, 144, 146, 148, 170, 174, 176, 180, or184.

In some embodiments, the enone reductase is capable of reducing(Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate with greater than 20 times the conversion rateof SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, the enone reductasecapable of reducing (Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate with greater than 20 times the conversion rateof SEQ ID NO:6 comprises an amino acid sequence that corresponds to thesequence of SEQ ID NO: 24, 28, 30, 54, 92, 100, 144, 148, 170, 180 or184.

In some embodiments, the engineered enone reductases can be used in amethod for converting/reducing(E)-2-(3,4-dimethyoxybenzylidene)-3-methylbutanal to2-(3,4-dimethyoxybenzyl)-3-methylbutanal, as illustrated below:

In some embodiments, the engineered enone reductase capable ofreducing/converting (E)-2-(3,4-dimethyoxybenzylidene)-3-methylbutanal to2-(3,4-dimethyoxybenzyl)-3-methylbutanal comprises an amino acidsequence with at least one of the following features: residuecorresponding to X5 is E; residue corresponding to X10 is P; residue atX28 is P; residue at X38 is S; residue at X40 is S or Y; residue at X44is Y; residue at X75 is L; residue at X83 is L, R, V, or I; residue atX117 is A, I, F, or S; residue at X124 is G or P; residue at X147 is G;residue at X154 is R; residue at X179 is R; residue at X248 is C;residue at X251 is A, C, E, L, R, S, V, I, or D; residue at X252 is H;residue at X294 is A; residue at X295 is T, G, or N; residue at X296 isG, R, A, S, E, K, I, or F; residue at X297 is G, F, K, Y, A, or G;residue at X302 is G; residue at X304 is K; residue at X305 is S;residue at X315 is P; residue at X330 is Y or R; residue at X333 is Q;residue at X369 is E; residue at X376 is I, T, A, or E; residue at X379is G; and/or residue at X397 is R. Certain of these engineered enonereductases are capable of converting greater than 90% of substrate(XVIII).

These engineered enone reductases include those comprising an amino acidsequence with at least one of the following features: residuecorresponding to X5 is E; residue corresponding to X28 is P; residuecorresponding to X44 is Y; residue corresponding to X83 is R, V, or I;residue corresponding to X147 is G; residue corresponding to X154 is R;residue corresponding to X179 is R; residue corresponding to X251 is A,E, V, or I; residue corresponding to X295 is T or N; residuecorresponding to X296 is A, S, E, K, or I; residue corresponding to X297is G, F, or A; residue corresponding to X305 is S; residue correspondingto X315 is P; residue corresponding to X330 is R; residue correspondingto X333 is A; residue corresponding to X339 is Q; residue correspondingto X369 is E; residue corresponding to X376 is I, I, A, or E; and/orresidue corresponding to X397 is R. Exemplary engineered reductasesinclude those corresponding to SEQ ID NOs: 138, 174, 40, 16, 18, 68, 76,96, 100, 106, 170, 176, 178, 182, 184, and 186.

In some embodiments, these engineered enone reductases comprise an aminoacid sequence with at least one of the following features: residuecorresponding to X10 is P; residue corresponding to X38 is S; residuecorresponding to X83 or R, V, or I; residue corresponding to X117 is F;residue corresponding to X147 is G; residue corresponding to X251 is V;residue corresponding to X296 is A; residue corresponding to X297 is F,Y, A, or G; residue corresponding to X315 is P; residue corresponding toX330 is R; residue corresponding to X333 is A; residue corresponding toX376 is I, K, A, or E; and/or residue corresponding to X379 is G.Exemplary engineered reductases include those corresponding to SEQ IDNOs: 142, 16, 18, 146, 178, 184, and 186. Certain of these engineeredenone reductases include those comprising an amino acid sequence with atleast one of the following features: residue corresponding to X83 is V;residue corresponding to X251 is V; residue corresponding to X297 is For A; residue corresponding to X333 is A; residue corresponding to X369is E; and/or residue corresponding to X376 is I, K, or A. Engineeredenone reductases of the present invention include those that are capableof converting 90% or more, 95% or more, and 99% or more of substrate XVto product XVI, and also include those capable of catalyzing theconversion to the R-enantiomer of XVI at a % ee of 80% or more, 90% ormore, 95%, or more, and 99% or more. In one embodiment, the engineeredenone reductase comprises an amino acid sequence comprising thefeatures: residue corresponding to X297 is F; residue corresponding toX369 is E; and residue corresponding to X376 is K. An exemplaryengineered enone reductase is illustrated by SEQ ID NO: 186.

Where the reference sequence is SEQ ID NO: 8, mutations that provideimproved activity and selectivity in one enantiomer of product (XVI)include Y83I or V, P296 A or I, S297 A or F, K369E, and Y376 A, I, or K.Mutations that provide improved properties activity and selectivity inthe other enantiomer of product (XVI) include T38S, W117A, N, or S, F251A, G, E, or V, S297F, and H330Y.

In some embodiments, the engineered enone reductase polypeptide iscapable of reducing the α,β unsaturated ketone that is an optionallysubstituted tetrahydronaphthaledione to the correspondinghexahydronaphthalendione. For example, the present invention providesengineered enone reductases that are capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione (XVIII) to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione (XIX) as follows:

In some embodiments, the engineered enone reductase is capable ofreducing 8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 2 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the engineered enone reductase capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione comprises an amino acidsequence with at least one of the following features: residuecorresponding to X38 is N; residue corresponding to X295 is T; residuecorresponding to X297 is F; residue corresponding to X251 is R; residuecorresponding to X252 is H; residue corresponding to X296 is A and X297is F; residue corresponding to X248 is C and X297 is G; residuecorresponding to X251 is L and X379 is G; residue corresponding to X296is K and X376 is A; residue corresponding to X296 is R and X330 is Y;residue corresponding to X83 is I and X251 is R; residue correspondingto X294 is A and X295 is G; residue corresponding to X10 is P, X297 is Gand X379 is G; residue corresponding to X40 is S, X302 is G and X330 isY; residue corresponding to X251 is I, X296 is S, and X297 is F; residuecorresponding to X251 is S, X297 is I and X358 is A; residuecorresponding to X83 is I, X251 is V and X295 is N; residuecorresponding to X251 is E, X330 is R and X376 is I; residuecorresponding to X38 is S, X83 is I, X251 is S and X296 is G; residuecorresponding to X296 is A, X297 is A, X330 is R and X376 is I; residuecorresponding to X296 is I, X297 is A, X333 is A and X376 is A; residuecorresponding to X251 is S, X296 is E, X297 is A and X311 is E; residuecorresponding to X251 is I, X296 is E, X297 is A and X376 is I; residuecorresponding to X251 is A, X296 is A, X297 is K and X399 is E; residuecorresponding to X147 is G, X296 is A, X297 is F, X330 is R and X376 isE; residue corresponding to X5 is E, X44 is Y, X83 is I, X251 is A, X295is N and X297 is G; residue corresponding to X240 is R, X251 is S, X259is G, X296 is Q and X297 is A; residue corresponding to X38 is S, X83 isI, X154 is R, X251 is V, X295 is T and X297 is F; residue correspondingto X38 is S, X83 is I, X251 is S, X295 is T, X296 is S, X297 is F andX384 is I; residue corresponding to X38 is S, X83 is I, X251 is S, X295is T, X296 is G and X297 is F; or residue corresponding to X38 is S, X83is I, X117 is F, X251 is S, X295 is N, X296 is G and X297 is F.

In some embodiments, the engineered enone reductase capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 2 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an amino acidsequence that corresponds to the sequence of SEQ ID NO: 12, 42, 68, 72,82, 86, 88, 98, 104, 106, 114, 118, 120, 122, 124, 126, 132, 136, 138,160, 162, 164, 166, 168, 170, 172, 176, 178, 180, 184, or 182.

In some embodiments, the engineered enone reductase is capable ofreducing 8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 5 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, theengineered enone reductase capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 5 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an amino acidsequence that corresponds to the sequence of SEQ ID NO: 82, 88, 104,114, 160, 164, 166 or 172.

In some embodiments, the engineered enone reductase is capable ofreducing 8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with greater than 10times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8. In someembodiments, the engineered enone reductase capable of reducing8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione with at least 10 timesthe conversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 164 or 172.

In some embodiments, the engineered enone reductase is capable ofreducing an optionally substituted cyclopentenone to an optionallysubstituted cyclopentanone. For example, the present invention providesengineered enone reductases of the present invention that are capable ofreducing 3-methylcyclohex-2-enone to 3-methylcyclohexanone, asillustrated below, with at least for 2 times the conversion rate of SEQID NO:6 or SEQ ID NO:8

In some embodiments, the enone reductase capable of reducing anoptionally substituted cyclohexenone to the corresponding cyclohexanone.For example, in some embodiments the engineered enone reductasepolypeptide is capable of reducing 3-methylcyclohex-2-enone to3-methylcyclohexanone, as illustrated below, with at least 1 times theconversion rate of SEQ ID NO:6 or SEQ ID NO:8:

In some embodiments, the enone reductase capable of reducing3-methylcyclohex-2-enone to 3-methylcyclohexanone with at least 1 timesthe conversion rate of SEQ ID NO: 6 or SEQ ID NO: 8 comprises an aminoacid sequence with at least one of the following features: residuecorresponding to X83 is V; residue corresponding to X297 is K; residuecorresponding to X83 is I and X124 is P; residue corresponding to X305is S and X376 is I; residue corresponding to X297 is F, X369 is E andX376 is K; or residue corresponding to X10 is P, X297 is G and X379 isG.

In some embodiments, the enone reductase capable of reducing3-methylcyclohex-2-enone to 3-methylcyclohexanone with at least 1 timesthe conversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 18, 46,132, 174, 186 or 140.

In some embodiments, the enone reductase is capable of reducing3-methylcyclohex-2-enone to 3-methylcyclohexanone with greater than 1.5times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8. In someembodiments, the enone reductase capable of reducing3-methylcyclohex-2-enone to 3-methylcyclohexanone with greater than 1.5times the conversion rate of SEQ ID NO:6 or SEQ ID NO:8 comprises anamino acid sequence that corresponds to the sequence of SEQ ID NO: 186or 132.

In some embodiments, the enone reductase is capable of reducing anoptionally substituted cyclopentenone to the correspondingcyclopentanone. For example, in some embodiments, the engineered enonereductase polypeptides of the present invention are capable of reducing2-methylcyclopente-2-none to 2-methylcyclopentanone as follows:

In certain embodiments, the engineered enone reductase polypeptide iscapable of reducing substrate (XXII) to a product that isenantiomerically enriched for either the (R) or (S) enantiomer ofproduct (XXIII). In some embodiments, the engineered enone reductasepolypeptide capable of reducing 2-methylcyclopente-2-none to2-methylcyclopentanone comprises an amino acid sequence with one of thefollowing features: residue corresponding to X38 is S, X83 is I, X117 isA, X251 is Y, X295 is T, X296 is F, and X297 is W; or residuecorresponding to X38 is S, X83 is I, X117 is I. X251 is A, X295 is N,X296 is F and X297 is W.

In some embodiments, the enone reductase capable of reducing2-methylcyclopente-2-none to 2-methylcyclopentanone comprises an aminoacid sequence that corresponds to the sequence of SEQ ID NO: 60 or 62.

Table 2 below provides exemplary chimeric enone reductase polypeptides,unless specified otherwise. In Table 2 below, each row lists two SEQ IDNOs, where the odd number refers to the nucleotide sequence that codesfor the amino acid sequence provided by the even number. In thedescription of the chimeric structure, the number preceding theparenthesis is the source of the segment and the numbers in parenthesisrefers to the segment from the first residue to the terminal residueposition. The chimeric structure is presented, from left to right, theamino to the carboxy terminus

TABLE 2 SEQ ID NO. CHIMERIC STRUCTURE OF POLYPEPTIDE 1/2 Old YellowEnzyme 1 Saccharomyces pastorianus (ERED 1) 3/4 Old Yellow Enzyme 2Saccharomyces cerevisiae (ERED 2) 5/6 Old Yellow Enzyme 3 Saccharomycescerevisiae (ERED 3) 189/1901(1-19)-3(20-59)-2(60-109)-3(110-141)-2(142-299)-3(300-400) 191/1923(1-59)-2(60-275)-3(276-400) 193/194 3(1-109)-2(110-250)-3-(251-400)195/196 3(1-80)-2(81-176)-3(177-337)-2(338-400) 197/1982(1-29)-3(30-103)-1(104-123)-2(124-400) 199/2002(1-19)-3(20-68)-2(69-103)-3(104-141)-2(142-176)-3(177-400) 201/2022(1-19)-3(20-250)-2(251-299)-3(300-400) 203/2043(1-68)-2(69-176)-3(177-240)-2(241-297)-3(298-400) 205/2063(1-311)-2(312-400) 207/2082(1-43)-3(44-56)-2(57-209)-3(210-260)-2(261-400) 209/2102(1-19)-3(20-127)-2(128-400) 211/2123(1-56)-1(57-74)-3(75-118)-2(119-400) 213/2143(1-43)-1(44-112)-2(113-400) 215/2162(1-19)-3(20-34)-1(35-72)-2(73-80)-3(81-109)-2(110-176)- 3(177-400) 7/81(1-17)-3(18-127)-2(128-275)-3(276-400)

The stabilities of the chimeric enone reductases with respect totemperature and/or isopropanol are presented in Table 3, where thepolypeptides were assayed for reduction of cyclohex-2-enone tocyclohexanone. Activity of the exemplary chimeric enone reductases onmethyl crotonate(methyl (E)-but-2-enoate) and 3-methyl-cyclohexanone arealso shown.

TABLE 3 ACTIVITY ON ACTIVITY ON SEQ ID 50% IPA 10% IPA at 20% IPA atMETHYL 3-METHYL NO. ON 30° C.¹ 40° C.¹ 40° C.¹ CROTONATE² CYCLOHEXENONE² 5/6 189/190 + + 191/192 + + 193/194 + + + 195/196 + 197/198 +199/200 + + 201/202 + + + 203/204 + + + 205/206 + 207/208 + +209/210 + + + 211/212 + + 213/214 + 215/216 + + 7/8 + + + + ¹Residualactivity = (activity after incubation with IPA/activity after incubationwithout IPA) * 100 + = Higher residual activity than OYE3 ²Relativeactivity calculated based on that of OYE3 + = Higher conversion thanOYE3

Table 4 below provides exemplary engineered enone reductase polypeptidesand, unless specified otherwise, the residue differences as compared tothe reference sequence of SEQ ID NO:8. As with Table 2, each row inTable 4 lists two SEQ ID NOs, where the odd number refers to thenucleotide sequence that codes for the amino acid sequence provided bythe even number. The column listing the residue differences is withrespect to the number of amino acid substitutions as compared to thechimeric ERED of SEQ ID NO:8.

TABLE 4 SEQ ID NO. RESIDUE DIFFERENCES FROM SEQ ID NO: 8 53/54 T38S;Y83I; W117A; F251D 55/56 T38S; Y83I; W117A; F251A 51/52 T38S; M40Y;Y83I; W117A 57/58 T38S; Y83I; W117I; K153E; F251W; D295T; P296F; S297Y61/62 T38S; Y83I; W117A; F251Y; D295T; P296F; S297W 59/60 T38S; Y83I;W117I; F251A; D295N; P296F; S297W 185/186 S297F; K369E; Y376K 131/132Q10P; S297G; S379G 173/174 Y305S; Y376I 17/18 Y83V 139/140 S297K 45/46Y83I; F124P 41/42 T38N  9/10 M40L; T294A 39/40 S315P; Y376T 123/124D295T 133/134 F75L; S297A 13/14 Y83L 137/138 S297F 119/120 N252H 129/130L119P 43/44 Y83I; F124G; E304K 177/178 P296I; S297A; V333A; Y376A 11/12M40S; E302G; H330Y 141/142 S297Y 161/162 P296A; S297F 135/136 Y248C;S297G 127/128 P296G 113/114 F251L; S379G 169/170 P296K; Y376A 125/126P296R; H330Y 179/180 P296A; S297A; H330R; Y376I 121/122 T294A; D295G 99/100 Y83I; P296S; S297F; Y376I; W397R 115/116 F251C 165/166 F251I;P296S; S297F 111/112 F251A 25/26 W117M 145/146 T38S; Y83I; S297Y 183.184E147G; P296A; S297F; H330R; Y376E 67/68 K5E; H44Y; Y83I; F251A; D295N;S297G 155/156 Y83E; W117I; V333A 167/168 F251S; S297I; V358A 71/72 Y83I;F251R 105/106 T38S; Y83I; K154R; F251V; D295T; S297F 73/74 Y83I; F251A109/110 Q148R; F251G 89/90 Y83I; F251V 75/76 Y83I; K179R; F251C; K339Q159/160 F251S; P296E; S297A; D311E 35/36 W117N 21/22 W117L 107/108 Y83I;F251S 175/176 F251I; P296E; S297A; Y376I 37/38 W117Q 69/70 Y83I; F251A;H330Y 117/118 F251R 171/172 F251A; P296A; S297K; K399E 33/34 W117E;Y386D 47/48 T38S; F75L; Y83I; W117I; S255P 163/164 K240R; F251S; E259G;P296Q; S297A 157/158 Y83I; W117I 149/150 Y83K; W117I 187/188 F251E153/154 T38S; Y83I; W117I 63/64 T38S; Y83I; W117L 23/24 W117A 87/88T38S; Y83I; F251S; D295T; P296S; S297F; T384I 49/50 T38S; Y83I; W117I;L119V 97/98 Y83I; F251V; D295N 95/96 Y83I; W117S; F251V; P296R; S297F181/182 F251E; H330R; Y376I 81/82 T38S; Y83I; F251S; P296G 77/78 T38S;Y83I; W117F; F251S 103/104 T38S; Y83I; F251S; D295T; P296G; S297F 27/28W117A; A122T 19/20 W117C 143/144 T38S; Y83I; W117A; H330Y 151/152 T38S;M40E; F75S; Y83I; W117I 147/148 T38S; W117A 83/84 T38S; Y83I; W117F;F251V; Y376I 85/86 T38S; Y83I; W117F; F251S; D295N; P296G; S297F 31/32W117I 65/66 T38S; Y83I; W117I; D295T; P296A 79/80 T38S; Y83I; W117L;N209D; F251S; Y376K; N400T 101/102 T38S; Y83I; W117N; F251V 29/30 W117V93/94 Y83I; W117N; D295T; P296G; S297F 91/92 L28P; Y83I; W117A; F251V15/16 Y83R

In some embodiments, an engineered enone reductase can comprise an aminoacid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to areference sequence listed in Table 3, with the proviso that the enonereductase has the thermal and/or solvent stability characteristicsdescribed herein. In some embodiments, the enone reductase polypeptidescan have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35,1-40, 1-45, 1-50, 1-55, or 1-60 residue differences, such as thosedescribed hereinabove, as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences. In some embodiments, the differences compriseconservative mutations.

In some embodiments, an engineered enone reductase can comprise an aminoacid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a referencesequence based on SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, or 188, with the proviso that the enone reductase amino acidsequence comprises any one of the set of mutations contained in any oneof the polypeptide sequences listed in Table 4. In some embodiments, theenone reductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other amino acid residue positions as compared to thereference sequence. In some embodiments, the number of differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24,26, 30, 35, 40, 45, 50, 55, or 60 residue differences at other aminoacid residues. In some embodiments, the differences compriseconservative mutations.

In some embodiments, the engineered enone reductase polypeptide cancomprise deletions of the naturally occurring enone reductasepolypeptides or deletions of the engineered enone reductasepolypeptides. In some embodiments, the engineered enone reductasepolypeptides can comprise deletions of the engineered enone reductasepolypeptides described herein. Thus, for each and every embodiment ofthe enone reductase polypeptides of the disclosure, the deletions cancomprise one or more amino acids, 2 or more amino acids, 3 or more aminoacids, 4 or more amino acids, 5 or more amino acids, 6 or more aminoacids, 8 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, up to 10% of the total number of amino acids, up to 20% of thetotal number of amino acids, or up to 30% of the total number of aminoacids of the enone reductase polypeptides, as long as the functionalactivity of the enone reductase activity is maintained. In someembodiments, the deletions can comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acid residues.In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 amino acids. In some embodiments, the deletions can comprisedeletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 22, 24, 26, 28, or 30 amino acid residues.

As described herein, the enone reductase polypeptides of the disclosurecan be in the form of fusion polypeptides in which the enone reductasepolypeptides are fused to other polypeptides, such as, by way of exampleand not limitation, antibody tags (e.g., myc epitope), purificationssequences (e.g., His tags), and cell localization signals (e.g.,secretion signals). Thus, the engineered enone reductase polypeptidescan be used with or without fusions to other polypeptides.

In some embodiments, the engineered enone reductases may be prepared andused in the form of cells expressing the enzymes, as crude extracts, oras isolated or purified preparations. The enone reductases may beprepared as lyophilizates, in powder form (e.g., acetone powders), orprepared as enzyme solutions. In some embodiments, the enone reductasescan be in the form of substantially pure preparations.

In some embodiments, the enone reductase polypeptides can be attached toa solid substrate. The substrate can be a solid phase, surface, and/ormembrane. A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of the substrate can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-stereomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered enonereductase enzyme can be targeted to a specific property of the enzyme.

Polynucleotides Encoding Enone Reductase Polypeptides

In another aspect, the present disclosure provides polynucleotidesencoding the engineered enone reductase polypeptides. Thepolynucleotides may be operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered enone reductases can be introduced into appropriate hostcells to express the corresponding enone reductase polypeptide.

Polynucleotides encoding engineered enone reductase polypeptides of thepresent invention include polynucleotides that hybridize under stringentconditions to a reference nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 7, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, and 215.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the engineered enzymesdisclosed herein. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the protein. Inthis regard, the present disclosure specifically contemplates each andevery possible variation of polynucleotides that could be made byselecting combinations based on the possible codon choices, and all suchvariations are to be considered specifically disclosed for anypolypeptide disclosed herein, including the amino acid sequencespresented in Tables 3 and 4.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells.

In certain embodiments, all codons need not be replaced to optimize thecodon usage of the enone reductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the enone reductase enzymes may containpreferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90%of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of thereference engineered enone reductase polypeptides described herein.Accordingly, in some embodiments, the polynucleotide encodes an aminoacid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 8.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 190.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 192.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 194.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 196.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 198.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 200.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 202.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 204.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 206.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 208.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 210.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 212.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 214.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding an enone reductase polypeptide with an amino acid sequence thathas at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to thepolypeptide comprising an amino acid corresponding to SEQ ID NO: 216.

In some embodiments, the polynucleotides encoding the enone reductasesare selected from SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, or 215.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more sequence identity at the nucleotide level to a referencepolynucleotide encoding the engineered enone reductases. In someembodiments, the reference polynucleotide is selected from SEQ ID NO: 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, or 215.

An isolated polynucleotide encoding the enone reductases herein may bemanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the isolated polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. Guidance is provided in Sambrook et al., 2001, MolecularCloning: A Laboratory Manual, 3^(rd) Ed., Cold Spring Harbor LaboratoryPress; and Current Protocols in Molecular Biology, Ausubel. F. ed.,Greene Pub. Associates, 1998, updates to 2006.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, E. coli trp operon,bacteriophage Streptomyces coelicolor agarase gene (dagA), Bacillussubtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylasegene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990, which is incorporated hereinby reference.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137, whichis incorporated herein by reference.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra, which is incorporated herein by reference.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the ERED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered enone reductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, ProcNatl Acad Sci. USA 75:1433, which is incorporated herein by reference).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTM™ expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

Host Cells for Expression of Enone Reductases

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved enone reductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe enone reductase enzyme in the host cell. Host cells for use inexpressing the ERED polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums and growthconditions for the above-described host cells are well known in the art.

Polynucleotides for expression of the enone reductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedenone reductase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lad repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 were isolated by subjecting the cells tochloramphenicol selection.

Methods of Generating Engineered Enone Reductase Polypeptides

In some embodiments, to make the engineered ERED polynucleotides andpolypeptides of the present disclosure, the naturally-occurring enonereductase enzyme that catalyzes the reduction reaction is obtained (orderived) from Saccharomyces pastorianis. In some embodiments, the parentpolynucleotide sequence is codon optimized to enhance expression of theenone reductase in a specified host cell.

The engineered enone reductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring enone reductases tomutagenesis and/or directed evolution methods, as discussed above. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767; U.S. Pat. No. 6,537,746; Stemmer, W. P. C., 1994,Nature 370:389-391; Crameri, A. et al., 1998, Nature 391:288-291; U.S.Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; and U.S. Pat. No.5,830,721. All references are incorporated herein by reference. Otherdirected evolution procedures that can be used include, among others,staggered extension process (StEP), in vitro recombination (Zhao et al.,1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al.,1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black etal., 1996, Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered enone reductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD⁺ or NADP⁺. In this reaction,the NADH or NADPH is consumed (oxidized) by the enone reductase as thereductase reduces an α,β unsaturated substrate to the correspondingsaturated compound. The rate of decrease of NADH or NADPH concentration,as measured by the decrease in absorbance or fluorescence, per unit timeindicates the relative (enzymatic) activity of the ERED polypeptide in afixed amount of the lysate (or a lyophilized powder made therefrom).Where the improved enzyme property desired is thermal or solventstability, enzyme activity may be measured after subjecting the enzymepreparations to a defined temperature and/or solvent and measuring theamount of enzyme activity remaining after heat treatments. Clonescontaining a polynucleotide encoding a enone reductase are thenisolated, sequenced to identify the nucleotide sequence changes (ifany), and used to express the enzyme in a host cell.

Where the sequence of the engineered enone reductase polypeptide isknown, the polynucleotides encoding the enzyme can be prepared bystandard solid-phase methods, according to known synthetic methods. Insome embodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Great American Gene Company, Ramona, Calif.,ExpressGen Inc. Chicago, Ill., Operon Technologies Inc., Alameda,Calif., and many others.

The present invention provides a method of producing an engineered enonereductase polypeptide, the method comprising culturing a host celltransformed with a polynucleotide encoding an engineered enone reductasepolypeptide of the present invention under conditions suitable for theexpression of the engineered enone reductase polypeptide. The engineeredenone reductase enzyme can optionally be recovered from the culturemedium or from the transformed and cultured cells. For example,engineered enone reductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the enone reductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theengineered enone reductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the enone reductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with an engineered polypeptide. The polypeptidemay be attached to a suitable carrier, such as BSA, by means of a sidechain functional group or linkers attached to a side chain functionalgroup. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette Guerin) and Corynebacterium parvum.

Methods of Using the Engineered Enone Reductase Enzymes and CompoundsPrepared Therewith

The present disclosure provides a method of reducing or converting anα,β unsaturated compound comprising (a) providing an α,β unsaturatedcompound selected from the group consisting of an α,β unsaturatedketone, and α,β unsaturated aldehyde, an α,β unsaturated nitrile, and anα,β unsaturated ester; and (b) contacting the α,β unsaturated compoundwith an engineered enone reductase polypeptide of the present inventionunder reaction conditions suitable for conversion of the α,β unsaturatedcompound to the corresponding saturated product compound (i.e.,saturated ketone, aldehyde, nitrile, or ester). Further illustrativeembodiments of this method are described hereinbelow.

In some embodiments, the enone reductases of the disclosure are capableof reducing the α,β unsaturated compound of formula (I) to the saturatedcompound of formula (II):

where

R¹ is H, OH, alkyl, CN, or halide;

R² is H, alkyl, aryl, aralkyl, or alkoxy;

R³ is H, alkyl, aryl, or C(O)R⁷, wherein R⁷ is H or alkyl;

R⁴ is CN, an aryl, C(O)R⁵, or C(O)OR⁶, wherein R⁵ is H or alkyl and R⁶is H or alkyl; and wherein R³ and R⁵ can form a ring, including a fusedring. In some embodiments, the ring can be substituted or unsubstituted.In the above, the alkyl group can be substituted or unsubstituted,branched or straight chain, and the aryl group substituted orunsubstituted. In some embodiments, the halide is Cl. Various substratesknown to be recognized by Old Yellow Enzymes are known in the art, suchas those described in Vas et al., 1995, Biochemistry 34:4246-4256, whichis incorporated herein by reference.

Accordingly, the engineered reductases can be used in a method forconverting the compound of formula (I) (“the substrate”) to the compoundof formula (II) (“the product), which method comprises incubating orcontacting the compound of formula (I) with an engineered enonereductase of the present disclosure under reaction conditions suitablefor the conversion of the substrate to the product of formula (II).

In some embodiments, the engineered enone reductases can be used toreduce the α,β unsaturated carbonyl compound of formula (V) to thesaturated carbonyl compound of formula (VI):

where,

R¹ is H, substituted or unsubstituted lower alkyl (e.g., C1-C4);

R² is substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy; and

R⁵ is substituted or unsubstituted alkyl.

The method can comprise incubating or contacting a compound of formula(V) with an enone reductase of the present disclosure under reactionconditions suitable for the conversion of compound (V) to the product offormula (VI).

In some embodiments, the enone reductases of the disclosure are capableof reducing the α,β unsaturated compound of formula (I) to the saturatedcompound of formula (II):

where

R¹ is H, OH, alkyl, CN, or halide;

R² is H or alkyl;

R⁴ is C(O)R⁵, and

R³ and R⁵ form a ring. In some embodiments, the ring comprises asubstituted or unsubstituted cycloalkyl. Exemplary cycloalkyl ringsinclude, cyclobutenyl, cyclopentenyl, cyclohexenyl; and the like,wherein the ring can be substituted or unsubstituted. In someembodiments, the ring comprises a fused ring. In the above, the alkylgroup can be substituted or unsubstituted, branched or straight chain.In some embodiments, the halide is Cl. In these embodiments, the methodcan comprise incubating or contacting the ring compound described in theforegoing with an enone reductase of the present disclosure undersuitable conditions to reduce the ring compound to the correspondingreduced ring product.

In some embodiments, the engineered enone reductases can be used in amethod for converting/reducing an optionally substituted cycloxenone tothe corresponding cyclohexanone, where the method comprises incubatingor contacting the cyclohexenone with an enone reductase of the presentinvention under reaction conditions suitable for converting or reducingthe optionally substituted cyclohexenone to the correspondingcyclohexanone. For example, the present invention provides a method forconverting/reducing 1-cyclohex-2-enone of formula (III) to thecyclohexanone of formula (IV):

In these embodiments, the method can comprise incubating or contacting acompound of formula (III) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (III) to the product of formula (IV).

In some embodiments, the engineered enone reductases can be used in amethod for converting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one(VII) to (2R,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (VIII) asfollows:

In these embodiments, the method can comprise incubating or contacting acompound of formula (VII) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (VII) to the product of formula (VIII).

In some embodiments, the engineered enone reductase can be used in amethod for converting (5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one(VII) to (2S,5S)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one (VIIIb) indiastereomeric excess, as illustrated in the following reaction:

In these embodiments, the method can comprise incubating or contacting acompound of formula (VII) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (VII) to the product of formula (VIIIb) in diastereomericexcess.

In some embodiments, the engineered enone reductase can be used in amethod for converting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one(R-carvone) (IX) to (2R,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one(X) with at least 90% diastereomeric excess, as illustrated below:

In these embodiments, the method can comprise incubating or contacting acompound of formula (IX) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (IX) to the product of formula (X) in diastereomeric excess.

In some embodiments, the engineered enone reductases can be used in amethod for converting (5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one(R-carvone) (IX) to (2S,5R)-2-methyl-5-prop-1-en-2-ylcyclohexan-1-one(Xb) in diastereomeric excess, as illustrated in the following reaction:

In these embodiments, the method can comprise incubating or contacting acompound of formula (IX) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (IX) to the product of formula (Xb) in diastereomeric excess.

The present invention also provides a method for reducing an optionallysubstituted arylalkenone to an optionally substituted arylalkanone. Insome embodiments, the method comprises incubating or contacting acompound of formula (XI) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XI) to (XII):

wherein for (XI) and (XII), R¹ and R² are each independently selectedfrom the group consisting of CN, C(O)R¹¹, C(O)OR¹¹, an alkyl (such as,for example, a lower alkyl), and H, wherein R¹¹ is selected from thegroup consisting of H and an alkyl (such as, for example, a loweralkyl), and wherein R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each independentlyselected from the group consisting of H, an alkyl (such as, for example,a lower alkyl), an alkoxy (such as, for example, a lower alkoxy), ahydroxyl, and a halide. Typically, only one of R¹ and R² is CN and onlyone of R¹ and R² is C(O)R¹¹ or C(O)OR¹¹. Usually, at least one of R¹ andR² is C(O)R¹¹ or C(O)OR¹¹. In some embodiments, R² is an alkyl, such as,for example, a lower alkyl (i.e., methyl, propyl, isopropyl, and thelike).

In some embodiments, the engineered enone reductases can be used in amethod for converting (Z)-ethyl 2-cyano-3-phenylbut-2-enoate (XIII) toethyl 2-cyano-3-phenylbutanoate (XIV) as follows:

In these embodiments, the method can comprise incubating or contacting acompound of formula (XIII) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XIII) to the product of formula (XIV) at a rate that isgreater conversion rate than the polypeptide of SEQ ID NO:6 or SEQ IDNO:8.

In some embodiments, the engineered enone reductases can be used in amethod for converting/reducing(E)-2-(3,4-dimethyoxybenzylidene)-3-methylbutanal (XV) to2-(3,4-dimethyoxybenzyl)-3-methylbutanal (XVI), as illustrated below:

In these embodiments, the method can comprise incubating or contacting acompound of formula (XV) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XV) to the product of formula (XVI).

In some embodiments, the engineered enone reductases can be used in amethod for converting8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione (XVIII) to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione (XIX) as follows:

In these embodiments, the method can comprise incubating or contacting acompound of formula (XVIII) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XVIII) to the product of formula (XIX) at a conversion ratethat is greater than the polypeptide of SEQ ID NO:6 or SEQ ID NO:8.

In some embodiments, the engineered enone reductases can be used in amethod for converting 3-methylcyclohex-2-enone (XX) to3-methylcyclohexanone (XXI), as illustrated below:

In these embodiments, the method can comprise incubating or contacting acompound of formula (XX) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XX) to the product of formula (XXI).

In some embodiments, the engineered enone reductases can be used in amethod for converting or reducing an optionally substitutedcyclopentenone to the corresponding cyclopentanone. For example, theengineered enone reductase polypeptides can be used in a method forconverting/reducing 2-methylcyclopente-2-none (XXII) to2-methylcyclopentanone (XXIII) as illustrated below:

In these embodiments, the method can comprise incubating or contacting acompound of formula (XXII) with an enone reductase of the presentdisclosure under reaction conditions suitable for the conversion ofcompound (XXII) to the product of formula (XXIII).

Since enone reductases use a cofactor, the engineered enone reductasescan be used in combination with a cofactor regenerating system in themethods for reducing the α,β unsaturated compounds. In some embodiments,the reduction by enone reductases results in the generation of NADP+from NADPH, and therefore any system capable of regenerating NADPH canbe used.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

Glucose dehydrogenase (GDH) are NAD⁺ or NADP⁺-dependent enzymes thatcatalyzes the conversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Equation (1), below, describes theglucose dehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by glucose.

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring are well known in the art and mayalso be readily identified using the assay described in Example 4 of PCTpublication WO 2005/018579, the disclosure of which is incorporatedherein by reference. Exemplary non-naturally occurring glucosedehydrogenases are provided in PCT publication WO 2005/018579 as SEQ IDNOS: 62, 64, 66, 68, 122, 124, and 126. The polynucleotide sequencesthat encode them are provided in PCT publication WO 2005/018579 as SEQID NOS: 61, 63, 65, 67, 121, 123, and 125, respectively. All of thesesequences are incorporated herein by reference. Additional non-naturallyoccurring glucose dehydrogenases that are suitable for use in the enonereductase-catalyzed reduction reactions disclosed herein are provided inU.S. application publication Nos. 2005/0095619 and 2005/0153417, thedisclosures of which are incorporated herein by reference.

Glucose dehydrogenases employed in the enone reductase-catalyzedreduction reactions described herein may exhibit an activity of at leastabout 10 μmol/min/mg and sometimes at least about 10² μmol/min/mg orabout 10³ μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in theassay described in Example 4 of PCT publication WO 2005/018579.

The enone reductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not completelyinactivate the enone reductase enzyme. Appropriate co-solvent systemscan be readily identified by measuring the enzymatic activity of thespecified engineered enone reductase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. Generally, the reduction can be carried outat a pH of about 10 or below, usually in the range of from about 5 toabout 10. In some embodiments, the reduction is carried out at a pH ofabout 9 or below, usually in the range of from about 5 to about 9. Insome embodiments, the reduction is carried out at a pH of about 8 orbelow, often in the range of from about 5 to about 8, and usually in therange of from about 6 to about 8. The reduction may also be carried outat a pH of about 7.8 or below, or 7.5 or below. Alternatively, thereduction may be carried out a neutral pH, i.e., about 7.

During the course of the reduction reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range by the addition of an acid ora base during the course of the reaction. Alternatively, the pH may becontrolled by using an aqueous solvent that comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,for example, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used.

When the glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (3) causes the pH of the reaction mixture to drop if theresulting aqueous gluconic acid is not otherwise neutralized. The pH ofthe reaction mixture may be maintained at the desired level by standardbuffering techniques, wherein the buffer neutralizes the gluconic acidup to the buffering capacity provided, or by the addition of a baseconcurrent with the course of the conversion. Combinations of bufferingand base addition may also be used. Suitable buffers to maintain desiredpH ranges are described above. Suitable bases for neutralization ofgluconic acid are organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like. The addition of abase concurrent with the course of the conversion may be done manuallywhile monitoring the reaction mixture pH or, more conveniently, by usingan automatic titrator as a pH stat. A combination of partial bufferingcapacity and base addition can also be used for process control.

When base addition is employed to neutralize gluconic acid releasedduring a enone reductase-catalyzed reduction reaction, the progress ofthe conversion may be monitored by the amount of base added to maintainthe pH. Typically, bases added to unbuffered or partially bufferedreaction mixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, the co-factor regenerating system can comprises aformate dehydrogenase. The terms “formate dehydrogenase” and “FDH” areused interchangeably herein to refer to an NAD⁺ or NADP⁺-dependentenzyme that catalyzes the conversion of formate and NAD⁺ or NADP⁺ tocarbon dioxide and NADH or NADPH, respectively. Formate dehydrogenasesthat are suitable for use as cofactor regenerating systems in the enonereductase-catalyzed reduction reactions described herein are well knownin the art and include both naturally occurring formate dehydrogenases,as well as non-naturally occurring formate dehydrogenases. Suitableformate dehydrogenases include those corresponding to SEQ ID NOS: 70(Pseudomonas sp.) and 72 (Candida boidinii) of PCT publication WO2005/018579, which are encoded by polynucleotide sequences correspondingto SEQ ID NOS: 69 and 71, respectively, of PCT publication 2005/018579,the disclosures of which are incorporated herein by reference. Formatedehydrogenases employed in the methods described herein, whethernaturally occurring or non-naturally occurring, may exhibit an activityof at least about 1 μmol/min/mg, sometimes at least about 10μmol/min/mg, or at least about 10² mmol/min/mg, up to about 10³mmol/min/mg or higher, and can be readily screened for activity in theassay described in Example 4 of PCT publication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂ ⁻),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa (pKa=3.7in water) formate is present as both HCO₂ ⁻ and HCO₂H in equilibriumconcentrations. At pH values above about pH 4, formate is predominantlypresent as HCO₂ ⁻. When formate is provided as formic acid, the reactionmixture is typically buffered or made less acidic by adding a base toprovide the desired pH, typically of about pH 5 or above. Suitable basesfor neutralization of formic acid include, but are not limited to,organic bases, for example amines, alkoxides and the like, and inorganicbases, for example, hydroxide salts (e.g., NaOH), carbonate salts (e.g.,NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basic phosphate salts (e.g.,K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂, Equation (2) below, describes the formatedehydrogenase-catalyzed reduction of NAD⁺ or NADP⁺ by formate.

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the pH of the reaction mixture may be maintained atthe desired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions.

The terms “secondary alcohol dehydrogenase” and “sADH” are usedinterchangeably herein to refer to an NAD⁺ or NADP⁺-dependent enzymethat catalyzes the conversion of a secondary alcohol and NAD⁺ or NADP⁺to a ketone and NADH or NADPH, respectively. Equation (3), below,describes the reduction of NAD⁺ or NADP⁺ by a secondary alcohol,illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the enone reductase-catalyzed reductionreactions described herein are well known in the art and include bothnaturally occurring secondary alcohol dehydrogenases, as well asnon-naturally occurring secondary alcohol dehydrogenases. Naturallyoccurring secondary alcohol dehydrogenases include known alcoholdehydrogenases from, Thermoanerobium brockii, Rhodococcus etythropolis,Lactobacillus kefir, and Lactobacillus brevis, and non-naturallyoccurring secondary alcohol dehydrogenases include engineered alcoholdehydrogenases derived therefrom. Secondary alcohol dehydrogenasesemployed in the methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment the secondaryalcohol is isopropanol. Suitable aryl-akyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, the resulting NAD⁺ orNADP⁺ is reduced by the coupled oxidation of the secondary alcohol tothe ketone by the secondary alcohol dehydrogenase. Some engineeredketoreductases also have activity to dehydrogenate a secondary alcoholreductant.

In carrying out embodiments of the enone reductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the enone reductase substrate.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

In carrying out the reduction reactions described herein, the engineeredenone reductase enzyme, and any enzymes comprising the optional cofactorregeneration system, may be added to the reaction mixture in the form ofthe purified enzymes, whole cells transformed with gene(s) encoding theenzymes, and/or cell extracts and/or lysates of such cells. The gene(s)encoding the engineered enone reductase enzyme and the optional cofactorregeneration enzymes can be transformed into host cells separately ortogether into the same host cell. For example, in some embodiments oneset of host cells can be transformed with gene(s) encoding theengineered enone reductase enzyme and another set can be transformedwith gene(s) encoding the cofactor regeneration enzymes. Both sets oftransformed cells can be utilized together in the reaction mixture inthe form of whole cells, or in the form of lysates or extracts derivedtherefrom. In other embodiments, a host cell can be transformed withgene(s) encoding both the engineered enone reductase enzyme and thecofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineered enonereductase enzyme and/or the optional cofactor regeneration enzymes, orcell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of enone reductase substrate employed. Thefollowing guidelines can be used to determine the amounts of enonereductase, cofactor, and optional cofactor regeneration system to use.Generally, substrates can be employed at a concentration of about 5 to300 grams/liter using from about 50 mg to about 5 g of enone reductaseand about 10 mg to about 150 mg of cofactor. Those having ordinary skillin the art will readily understand how to vary these quantities totailor them to the desired level of productivity and scale ofproduction. Appropriate quantities of optional cofactor regenerationsystem may be readily determined by routine experimentation based on theamount of cofactor and/or enone reductase utilized.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, enone reductase, and enone reductase substrate may be addedfirst to the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, enone reductase, and cofactormay be added and mixed into the aqueous phase first. The organic phasemay then be added and mixed in, followed by addition of the enonereductase substrate. Alternatively, the enone reductase substrate may bepremixed in the organic phase, prior to addition to the aqueous phase

Suitable conditions for carrying out the enone reductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineered enonereductase enzyme and substrate at an experimental pH and temperature anddetecting product, for example, using the methods described in theExamples provided herein.

The enone reductase catalyzed reduction is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or near complete, reduction of substrate is obtained.Reduction of substrate to product can be monitored using known methodsby detecting substrate and/or product. Suitable methods include gaschromatography, HPLC, and the like. Conversion yields of the reductionproduct generated in the reaction mixture are generally greater thanabout 50%, may also be greater than about 60%, may also be greater thanabout 70%, may also be greater than about 80%, may also be greater than90%, and are often greater than about 97%.

EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Generation of Chimeric Enone Reductases

The chimeric enone reductases were generated by standard domainshuffling methods as described in Stemmer, W. P. C., 1994, “Rapidevolution of a protein in vitro by DNA shuffling,” Nature 370:389-391;Stemmer, W. P. C., 1994, “DNA Shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution,” Proc NatlAcad Sci. USA 91:10751; Crameri, A. et al., 1998, “DNA shuffling of afamily of genes from diverse species accelerates directed evolution.”Nature 391:288-291; U.S. Pat. No. 5,605,793 (Stemmer); U.S. Pat. No.5,811,238 (Stemmer and Crameri); and U.S. Pat. No. 5,830,721 (Stemmerand Crameri). All references are incorporated herein by reference. Theenone reductases polynucleotides used to generate the chimeras were thegenes encoding Old Yellow Enzyme 1 from Saccharomyces pastorianus, OldYellow Enzyme 2 from Saccharomyces cerevisiae, and Old Yellow Enzyme 3from Saccharomyces cerevisiae, and presented herein as SEQ ID NO:1, SEQID NO:3, and SEQ ID NO:5, respectively.

As noted above, polynucleotides encoding engineered enone reductases ofthe present invention may also be cloned into any suitable vector, suchas vector pCK110900 (depicted in FIG. 3 in U.S. Patent ApplicationPublication 20060195947, which is incorporated herein by reference) forexpression in E. coli W3110.

Production of Enone Reductase Powders Shake Flask Procedure

A single microbial colony of E. coli containing a plasmid encoding anenone reductase of interest is inoculated into 50 ml Luria-Bertani brothcontaining 30 μg/ml chloramphenicol and 1% glucose. Cells are grownovernight (at least 16 hrs) in an incubator at 30° C. with shaking at250 rpm. The culture is diluted into 250 ml Terrific Broth (12 g/Lbacto-tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/ml chloramphenicol, in a1 liter flask to an optical density at 600 nm (OD₆₀₀) of 0.2 and allowedto grow at 30° C. Expression of the enone reductase gene is induced byaddition of iso-propyl-β-D-thiogalactoside (IPTG) to a finalconcentration of 1 mM when the OD₆₀₀ of the culture is 0.6 to 0.8 andincubation is then continued overnight (at least 16 hrs). Cells areharvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet is resuspended with an equalvolume of cold (4° C.) 100 mM triethanolamine (chloride) buffer, pH 7.5,and harvested by centrifugation as above. The washed cells areresuspended in two volumes of the cold triethanolamine (chloride) bufferand passed through a French Press twice at 12,000 psi while maintainedat 4° C. Cell debris is removed by centrifugation (9000 rpm, 45 min, 4°C.). The clear lysate supernatant is collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry powder of crudeenone reductase enzyme. Alternatively, the cell pellet (before or afterwashing) may be stored at 4° C. or −80° C.

Production of Enone Reductases Fermentation Procedure

Bench-scale fermentations are carried out at 30° C. in an aerated,agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/L ammoniumsulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogenphosphate trihydrate, 6.25 g/L of potassium dihydrogen phosphate, 6.2g/L of Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, and8.3 ml/L of a trace element solution containing 2 g/L of calciumchloride dihydrate, 2.2 g/L of zinc sulfate septahydrate, 0.5 g/Lmanganese sulfate monohydrate, 1 g/L cuprous sulfate heptahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate).The fermentor is inoculated with a late exponential culture of E. coliW3110 containing a plasmid encoding the enone reductase gene of interest(grown in a shake flask as described in Example 2) to a starting OD₆₀₀of 0.5 to 2.0. The fermentor is agitated at 500-1500 rpm and air issupplied to the fermentation vessel at 1.0-15.0 L/min to maintain adissolved oxygen level of 30% saturation or greater. The pH of theculture is maintained at 7.0 by addition of 20% v/v ammonium hydroxide.Growth of the culture is maintained by addition of a feed solutioncontaining 500 g/L cerelose, 12 g/L ammonium chloride and 10.4 g/Lmagnesium sulfate heptahydrate. After the culture reaches an OD600 of50, expression of enone reductase is induced by addition ofisopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mMand fermentation continued for another 14 hours. The culture is thenchilled to 4° C. and maintained at that temperature until harvested.Cells are collected by centrifugation at 5000 G for 40 minutes in aSorval RC12BP centrifuge at 4° C. Harvested cells are used directly inthe following downstream recovery process or they may be stored at 4° C.or frozen at −80° C. until such use.

The cell pellet is resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 6.8, at 4° C. to each volume of wet cell paste.The intracellular enone reductase is released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate is cooled to 4° C. immediately after disruption. A solutionof 10% w/v polyethyleneimine, pH 7.2, is added to the lysate to a finalconcentration of 0.5% w/v and stirred for 30 minutes. The resultingsuspension is clarified by centrifugation at 5000 G in a standardlaboratory centrifuge for 30 minutes. The clear supernatant is decantedand concentrated ten-fold using a cellulose ultrafiltration membranewith a molecular weight cut off of 30 kD. The final concentrate isdispensed into shallow containers, frozen at −20° C. and lyophilized topowder. The enone reductase powder is stored at −80° C.

Analytical Methods Conversion of Unsaturated Enone Substrate toSaturated Ketone Product and Stereopurity of Product

Achiral GC method to determine conversion of methyl crotonate to methylbutanoate: Reduction of methyl crotonate to methyl butanoate isdetermined using an Agilent 6890 GC-FID equipped with a 30 m×0.32 mm(0.25 μm film) Agilent HP-5 column using He as carrier gas with thefollowing temperature program: 20 psi, 60° C. for 3 min. Retentiontimes: methyl butanoate: 3.1 min; methyl crotonate: 3.6 min.

Achiral GC method to determine conversion of(S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone to (2R or 2S,5S)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone and diastereomeric purityof 2-methyl-5-(prop-1-en-2-yl)cyclohexanone: Reduction of(S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone to (2R or 2S,5S)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone is determined using anAgilent 6890 GC-FID equipped with a 30 m×0.32 mm (0.25 μm film) AgilentHP-5 column using He as carrier gas with the following temperatureprogram: 25 psi, 125° C. for 3.25 min. Retention times: (2S,5S)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone: 2.6 min;(2R,5S)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone: 2.7 min;(S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone: 3.1 min.

Achiral GC method to determine conversion of(R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone to (2R or 2S,5R)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone and diastereomeric purityof 2-methyl-5-(prop-1-en-2-yl)cyclohexanone: Reduction of(R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone to (2R or 2S,5S)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone is determined using anAgilent 6890 GC-FID equipped with a 30 m×0.32 mm (0.25 μm film) AgilentHP-5 column using He as carrier gas with the following temperatureprogram: 25 psi, 125° C. for 3.25 min. Retention times:(2R,5R)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone: 2.5 min;(2S,5R)-2-methyl-5-(prop-1-en-2-yl)cyclohexanone: 2.6 min;(S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone: 2.9 min.

Achiral GC method to determine conversion of (E/Z)-ethyl2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoate and thediastereomeric purity of ethyl 2-cyano-3-phenylbutanoate: Reduction ofethyl 2-cyano-3-phenylbut-2-enoate to ethyl 2-cyano-3-phenylbutanoate isdetermined using an Agilent 6890 GC-FID equipped with a 30 m×0.32 mm(0.25 μm film) Agilent HP-5 column using He as carrier gas with thefollowing temperature program: 20 psi, 170° C. for 5 min. Retentiontimes: diastereomer-1 of ethyl 2-cyano-3-phenylbutanoate: 3.3;diastereomer-2 of ethyl 2-cyano-3-phenylbutanoate: 3.4 min; (E orZ)-ethyl 2-cyano-3-phenylbut-2-enoate: 4.1 min; (E or Z)-ethyl2-cyano-3-phenylbut-2-enoate: 4.9 min.

Achiral GC method to determine conversion of8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione and the diastereomericpurity of 8a-methylhexahydronaphthalene-1,6(2H,7H)-dione: Reduction of8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione to8a-methylhexahydronaphthalene-1,6(2H,7H)-dione is determined using anAgilent 6890 GC-FID equipped with a 30 m×0.32 mm (0.25 μm film) AgilentHP-5 column using He as carrier gas with the following temperatureprogram: 20 psi, 165° C. for 4 min. Retention times: diastereomer-1 of8a-methylhexahydronaphthalene-1,6(2H,7H)-dione: 3.2 min; diastereomer-2of 8a-methylhexahydronaphthalene-1,6(2H,7H)-dione: 3.5 min;8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione: 3.8 min.

Chiral GC method to determine conversion of 3-methylcyclohexenone to3-methylcyclohexanone and the enantiopurity of 3-methylcyclohexanone:Reduction of 3-methylcyclohexenone to 3-methylcyclohexanone isdetermined using an Agilent 6890 GC-FID equipped with a 30 m×0.25 mm(0.25 μm film) Restek chiral Rt-βDex_(sm) column using He as carrier gaswith the following temperature program: 18 psi, 110° C. for 8 min; 25°C.min⁻¹, 150° C. for 1.4 min. Retention times:(R)-3-methylcyclohexanone: 7.1 min; (S)-3-methylcyclohexanone: 7.3 min;3-methylcyclohexenone: 10.4 min.

Chiral GC method to determine conversion of 2-methyl-2-pentenal to2-methylpentanol and the enantiopurity of 2-methylpentanol: Reduction of2-methyl-2-pentenal to 2-methylpentanol is determined using an Agilent6890 GC-FID equipped with a 30 m×0.25 mm (0.25 μm film) SupelcoBetadex-225 column using He as carrier gas with the followingtemperature program: 12 psi, 80° C. for 9.5 min; 70° C.min⁻¹, 150° C.for 2 min. Retention times: (R)-2-methylpentanol: 8.0 min;(S)-2-methylpentanol: 8.1 min; 2-methyl-2-pentenal: 11.9 min.

Chiral GC method to determine conversion of 2-methyl-2-pentenal to2-methylpentanol and the enantiopurity of 2-methylpentanol: Reduction of2-methyl-2-pentenal to 2-methylpentanol is determined using an Agilent6890 GC-FID equipped with a 30 m×0.25 mm (0.25 μm film) SupelcoBetadex-225 column using He as carrier gas with the followingtemperature program: 12 psi, 80° C. for 9.5 min; 70° C.min⁻¹, 150° C.for 2 min. Retention times: (R)-2-methylpentanol: 8.0 min;(S)-2-methylpentanol: 8.1 min; 2-methyl-2-pentenal: 11.9 min.

Chiral HPLC method to determine conversion of(E)-2-(3,4-dimethoxybenzylidene)-3-methylbutanal to give (R orS)-2-(3,4-dimethoxybenzyl)-3-methylbutanal and the enantiomeric purityof (R or S)-2-(3,4-dimethoxybenzyl)-3-methylbutanal: Reduction of(E)-2-(3,4-dimethoxybenzylidene)-3-methylbutanal to give (R orS)-2-(3,4-dimethoxybenzyl)-3-methylbutanal is determined using anAgilent 1100 HPLC equipped with a Chiralcel OJ-H column (15 cm length,4.6 mm diameter) using 85:15 heptane/isopropanol as eluent at a flowrate of 1 ml/min; and at a column temperature of 30° C.; UV=225 nm.Retention times: enantiomer-1 of2-(3,4-dimethoxybenzyl)-3-methylbutanal: 3.7 min; enantiomer-2 of2-(3,4-dimethoxybenzyl)-3-methylbutanal: 4.7 min;(E)-2-(3,4-dimethoxybenzylidene)-3-methylbutanal: 5.6 min.

Cell Selection, Growth, and Induced Expression of Enone ReductasePolypeptides (Variant Enzymes)

Individual colonies are robotically picked with a Q-Bot™ instrument(Genetix, USA Inc., Boston, Mass.) to 180 μL Luria-Bertani (LB) brothcontaining 1% glucose and 30 μg/mL chloramphenicol (CAM) in a 96 wellNUNC® plate (Nalge Nunc International, Rochester N.Y.). The plate (the“masterplate”) is sealed with AirPore tape (Qiagen, Valencia Calif.) anda lid, and incubated overnight at 30° C. at 250 rpm (2 inch throw) at85% relative humidity. Masterplates are subcultured by inoculating a 10μL aliquot from each well into a well of a Costar® deep well plate(Corning®, Acton Mass.) containing 390 μL Terrific Broth, pH 7.0,supplemented with 30 μg/ml chloramphenicol (CAM). The inoculated Costar®deep well plates are incubated for 2.5 hours at 30° C., 85% relativehumidity, at 250 rpm on a shaker with a 2 inch throw. The inducer IPTGis then added to each well to a final concentration of 1 mM andincubation continued for an additional 18 hours. Cells are harvested bycentrifuging the Costar® deep well plates at 4000 rpm for 10 minutes,discarding the supernatant. Generally, the pellets are frozen for onehour before lysis.

Glycerol is added to the wells of the masterplate to a finalconcentration of 20%. Masterplates are then stored at −80° C.

Screen for Enone Reductases Capable of Reducing Cyclohexenone in thePresence of NADPH Yielding NADP⁺ and Cyclohexanone

Recombinant E. coli colonies carrying a gene encoding enone reductaseare picked using a Q-Bot® robotic colony picker (Genetix USA, Inc.,Boston, Mass.) into 96-well shallow well microtiter plates containing180 μL Luria-Bertani (LB), 1% glucose and 30 μg/mL chloramphenicol(CAM). Cells are grown overnight at 30° C. with shaking at 200 rpm. A 10μL aliquot of this culture is then transferred into 96-deep well platescontaining 390 μL Terrific Broth (TB) and 30 μg/mL CAM. After incubationof the deep-well plates at 30° C. with shaking at 250 rpm for 2.5 hours,recombinant gene expression within the cultured cells is induced byaddition of IPTG to a final concentration of 1 mM. The plates are thenincubated at 30° C. with shaking at 250 rpm for 18 hrs.

Cells are pelleted by centrifugation (4000 RPM, 10 min, 4° C.),resuspended in 200 μL lysis buffer and lysed by shaking at roomtemperature for 2 hours. The lysis buffer contains 100 mM triethylamine(chloride) buffer or 100 mM sodium phosphate buffer, pH 7.5, 1 mg/mLlysozyme, and 500 μg/mL polymixin B sulfate (PMBS). After sealing theplates with aluminum/polypropylene laminate heat seal tape (Velocity 11(Menlo Park, Calif.), Cat#06643-001), they are shaken vigorously for 2hours at room temperature. Cell debris is collected by centrifugation(4000 RPM, 10 min., 4° C.) and the clear supernatant is assayed directlyor stored at 4° C. until use.

In this assay, 20 μl of sample (diluted in 100 mMtriethanolamine(chloride) buffer or 100 mM sodium phosphate buffer, atthe same pH as the lysis buffer) is added to 180 μl of an assay mixturein a well of 96-well black microtiter plates. Assay buffer consists of100 mM triethanolamine (chloride) buffer or 100 mM sodium phosphatebuffer, pH 7.5, 0.1 mg/ml cyclohexenone and 0.1 mg/ml NADPH. Thereaction is followed by measuring the reduction in fluorescence of NADPHas it is converted to NADP⁺ using a Flexstation® instrument (MolecularDevices, Sunnyvale, Calif.). NADPH fluorescence is measured at 445 nmupon excitation at 330 nm. For residual activity determination, samplesof lysates were preincubated at 30° C. or 40° C. in the presence of 10,20 or 50% IPA or additional co-solvents prior to addition to the assaymixture and compared to the corresponding lysate diluted with bufferonly. Activity measurement of lysate preincubated in co-solvent dividedby activity measurement of lysate preincubated in buffer yields theresidual activity.

Assay of Enone Reductase Activity Stereoselective Reduction of Enones orEnoates to Corresponding Products

Cell lysis: Cell pellets (collected in the wells of a microtiter plate)are lysed by addition of 200 μL lysis buffer (0.5 mg/ml PMBS, 1 mg/mllysozyme, 100 mM triethylamine (chloride) or 100 mM sodium phosphate, pH7.5, to each well. The plates are sealed, shaken vigorously for twohours at room temperature, and then centrifuged at 4000 rpm for 20minutes at 4° C. The supernatants are recovered and stored at 4° C.until assayed.

Enzymatic reduction reaction using glucose dehydrogenase (GDH), glucosefor cofactor recycling: For substrates with ketone or aldehydefunctionality (e.g., 3-methyl cyclohexenone), 100 μl of cell lysate istransferred to a deep well plate (Costar #3960) (Corning®, Acton Mass.)containing 150 IA of assay mix (per 100 ml: 83 ml 100 mM sodiumphosphate (pH 7.5), 83 mg Na-NADP (Oriental Yeast, Andover, Mass.), 835mg enone substrate, 334 mg appropriate glucose dehydrogenase, 2 gglucose, and 17 ml tetrahydrofuran). After sealing the plates withaluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park,Calif.), Cat#06643-001), reactions are run for 18-24 hrs at temperaturesranging from ambient to 30° C. Reactions are quenched by the addition of1 ml MTBE. Plates are resealed, shaken for 5 min, and the organic andaqueous layer separated by centrifugation (4000 rpm, 5 min, at ambienttemperature). 200 μl of the organic layer of each well is transferredinto the wells of a new shallow-well polypropylene plate (Costar #3365)(Corning®, Acton Mass.). After resealing the plates, samples aresubjected to GC analysis as described in example 4.

Enzymatic reduction reaction using ketoreductase, isopropanol forcofactor recycling: For substrates without ketone or aldehydefunctionality (e.g., methyl crotonate), 100 μl of cell lysate istransferred to a deep well plate (Costar #3960) (Corning®, Acton Mass.)containing 150 μl of assay mix (per 100 ml: 67 ml 100 mMtriethanolamine(chloride) (pH 7.5), 83 mg Na-NADP (Oriental Yeast,Andover, Mass.), 835 mg enoate substrate, 167 mg of appropriateketoreductase and 33 ml isopropyl alcohol). After sealing the plateswith aluminum/polypropylene laminate heat seal tape (Velocity 11 (MenloPark, Calif.), Cat#06643-001), reactions are run for 18-24 hrs attemperatures ranging from ambient to 30° C. Reactions are quenched bythe addition of 1 ml MTBE. Plates are resealed, shaken for 5 min, andthe organic and aqueous layer separated by centrifugation (4000 rpm, 5min, at ambient temperature). 200 μl of the organic layer of each wellis transferred into the wells of a new shallow-well polypropylene plate(Costar #3365) (Corning®, Acton Mass.). After resealing the plates,samples are subjected to GC analysis as described in example 4.

Table 5 provides the activities of exemplary enone reductases for(5S)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (S-carvone) and(5R)-2-methyl-5-prop-1-en-2-ylcyclohex-2-en-1-one (R-carvone) and theproducts generated by the enone reductases.

TABLE 5 S-CARVONE R-CARVONE SEQ ID NO: % CONV^(A) % DE^(B) % CONV.^(C) %DE^(D) 53/54 + S ++ N.D. 55/56 S N.D. 51/52 S + N.D. 57/58 RACEMIC N.D.61/62 RACEMIC N.D. 59/60 S N.D. 185/186 ++++ 131/132 + R + ++ 173/174 R+++ 17/18 ++ R + + +++ 139/140 + R + +++ 45/46 +++ R ++ + +++ 7/8 +R + + +++ 41/42 R + +++  9/10 ++ R + + +++ 39/40 R S +++ 123/124 + R ++133/134 + R + + +++ 13/14 ++ R ++ + +++ 137/138 R + +++ 119/120 + R ++++ 129/130 ++++ R ++ + ++ 43/44 +++ R + + +++ 177/178 R ++ 11/12 + R+++ 141/142 R + + +++ 161/162 ++ R + + +++ 135/136 + R +++ 127/128 ++R + + +++ 113/114 +++ R ++ +++ 169/170 R +++ 125/126 ++ R + +++ 179/180R ++ 121/122 ++ R + + +++ 99/100 R ++ 115/116 +++ R ++ + +++ 165/166 +R + +++ 111/112 +++ R ++ +++ 25/26 RACEMIC +++ 145/146 R + + +++ 183/183R ++ 67/68 ++++ R ++ + +++ 155/156 +++ S RACEMIC 167/168 + R + +++ 71/72++++ R ++ + +++ 105/106 + R ++ +++ 73/74 +++ R ++ + +++ 109/110 ++ R +++++ 89/90 ++++ R ++ + +++ 75/76 ++++ R ++ + +++ 159/160 R + ++ 35/36RACEMIC +++ 21/22 + R + + +++ 107/108 +++ R ++ + +++ 175/176 R ++ 37/38S ++ 69/70 +++ R ++ + +++ 117/118 +++ R + + +++ 171/172 ++++ R ++ + +++33/34 RACEMIC ++++ 47/48 ++++ S +++ RACEMIC 163/164 + R + ++ 157/158 +++S + + 149/150 + Racemic ++ 187/188 R ++ 153/154 ++++ S +++ racemic 63/64++++ S + + + 23/24 S +++ racemic 87/88 ++ R + +++ 49/50 ++++ S +++ +97/98 ++++ R ++ + +++ 95/96 + R ++ + +++ 181/182 R ++ 81/82 +++ R ++ ++++ 77/78 + R + +++ 103/104 ++ R ++ +++ 27/28 S ++ + 19/20 S + S+143/144 +++ S +++ S+ 151/152 ++++ S +++ + 147/148 +++ S +++ S+ 83/84 +85/86 ++ R ++ + +++ 31/32 + S +++ 65/66 + S ++ 79/80 Racemic +++ 101/102++ R + +++ 29/30 S ++ + 93/94 R +++ 91/92 S + 15/16 +++ R ++ + +++ ^(A)+= 100-149% of parent (SEQ ID NO: 8) ++ = 150-199% of parent +++ =200-250% of parent ++++ = >250% of parent ^(B)+++ = >95% ee ++ = 90-95%ee + = 80-90% ee None = 30-80% ee Racemic = <30% ee S = (2S,5S) productR = (2R,5S) product ^(C)+ = >100% of parent (SEQ ID NO: 8) ^(D)+++= >95% ee ++ = 90-95% EE + = 80-90% EE None = 30-80% ee Racemic = <30%ee S = (2S,5R) Product

Table 6 provides the activities of exemplary enone reductases onconversion of (Z)-ethyl 2-cyano-3-phenylbut-2-enoate to ethyl2-cyano-3-phenylbutanoate.

TABLE 6 ethyl 2-cyano-3-phenylbut-2-enoate SEQ ID NO: % conv.^(a) 53/54++++ 55/56 ++ 51/52 + 57/58 + 61/62 + 185/186 + 131/132 + 173/174 +++17/18 + 139/140 + 45/46 + 7/8 +  9/10 + 39/40 ++ 123/124 + 133/134 +13/14 + 137/138 + 129/130 + 43/44 +++ 177/178 ++ 11/12 + 141/142 +161/162 + 135/136 + 127/128 ++ 113/114 + 169/170 ++++ 125/126 + 179/180++++ 121/122 +  99/100 ++++ 115/116 + 165/166 + 25/26 ++ 145/146 +++183/183 ++++ 67/68 + 155/156 ++ 71/72 + 105/106 + 73/74 + 109/110 +89/90 75/76 + 159/160 + 35/36 ++ 21/22 ++ 107/108 + 175/176 +++ 37/38 +69/70 + 117/118 + 171/172 + 33/34 + 47/48 +++ 163/164 + 157/158 ++149/150 + 153/154 ++ 63/64 + 23/24 ++++ 87/88 + 49/50 +++ 97/98 + 95/96++ 181/182 ++ 81/82 ++ 77/78 + 103/104 + 27/28 ++++ 19/20 +++ 143/144++++ 151/152 ++ 147/148 ++++ 83/84 + 85/86 + 31/32 + 65/66 + 101/102 +29/30 ++++ 93/94 +++ 91/92 ++++ 15/16 + ^(a)+ = 100-500% of parent (SEQID NO: 8) ++ = 500-1000% of parent +++ = 1000-2000% of parent ++++= >2000% of parent

Table 7 shows the activities of exemplary enone reductases on theconversion of 8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dioneto 8a-methylhexahydronaphthalene-1,6(2H,7H)-dione.

TABLE 7 8a-methyl-3,4,8,8a-tetrahydronaphthalene-1,6(2H,7H)-dione SEQ IDNO: % conv.^(a) 185/186 + 131/132 ++ 173/174 + 139/140 + 7/8 + 41/42 +++ 9/10 + 39/40 + 123/124 ++ 133/134 + 137/138 ++ 119/120 ++ 129/13043/44 + 177/178 ++ 11/12 ++ 141/142 + 161/162 ++ 135/136 ++ 127/128 +113/114 +++ 169/170 ++ 125/126 ++ 179/180 ++ 121/122 ++  99/100 +115/116 + 165/166 +++ 111/112 + 145/146 + 183/183 ++ 67/68 ++ 167/168 ++71/72 ++ 105/106 ++ 75/76 + 159/160 +++ 107/108 + 175/176 ++ 117/118 ++171/172 ++++ 33/34 + 163/164 ++++ 187/188 + 87/88 +++ 97/98 ++ 181/182++ 81/82 +++ 77/78 + 103/104 +++ 143/144 + 85/86 ++ ^(a)+ = 100-200% ofparent (SEQ ID NO: 8) ++ = 200-500% of parent +++ = 500-1000% of parent++++ = >1000% of parent

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed:
 1. A method of reducing an α,β unsaturated compound,the method comprising: (a) providing: (i) an α,β unsaturated compoundselected from the group consisting of an α,β unsaturated ketone, an α,βunsaturated aldehyde, an α,β unsaturated nitrile, and an α,β unsaturatedester; and (ii) an enone reductase polypeptide comprising an amino acidsequence that is at least 80% identical to a SEQ ID NO:8, wherein theresidue corresponding to X251 is A, C, R, E, D, W, Y, S, V, G, L, or I;and (b) contacting the α,β unsaturated compound with the enone reductasepolypeptide under reaction conditions suitable for conversion of the α,βunsaturated compound to the corresponding saturated product compoundselected from the group consisting of a saturated ketone, a saturatedaldehyde, a saturated nitrile, and a saturated ester.
 2. The method ofclaim 1, wherein the α,β unsaturated compound has the structure offormula (I) and the saturated product compound has the structure offormula (II):

wherein for (I) and (II), R¹ is H, OH, alkyl, CN, or halide; R² is H,alkyl, aryl, aralkyl or alkoxy; R³ is H, alkyl, aryl, or C(O)R⁷, whereinR⁷ is H or alkyl; R⁴ is CN, aryl, C(O)R⁵, or C(O)OR⁶, wherein R⁵ is H oralkyl and R⁶ is H or alkyl; and wherein R³ and R⁵ can form a ring orfused ring.
 3. The method of claim 1, in which the α,β unsaturatedcompound is selected from the group consisting of 5-carvone, R-carvone,3-methylcyclohex-2-enone, methyl crotonate, and 2methyl-cyclpent-2-enone.
 4. The method of claim 1, wherein the reactionis carried out in the presence of a cofactor regenerating system.
 5. Themethod of claim 4, wherein the co-factor regenerating system is aketoreductase and an alkanol.
 6. The method of claim 5, wherein thealkanol is isopropanol.
 7. The method of claim 1, wherein said enonereductase polypeptide comprises an amino acid sequence that is at least80% identical to the reference sequence set forth in SEQ ID NO:8,wherein the amino acid sequence further comprises one or more residuedifferences as compared to the reference sequence, and the residuecorresponding to X117 is a cysteine or nonpolar, aliphatic, basic,acidic, polar, or aromatic residue, selected from C, L, A, M, V, I, N,Q, E, F, and S, and/or the residue corresponding to X38 is N or S,wherein said enone reductase polypeptide has enone reductase activity.8. The method of claim 1, wherein said enone reductase polypeptidefurther comprises a substitution set selected from: the residuecorresponding to X38 is S and X117 is A; the residue corresponding toX83 is K and X117 is I; the residue corresponding to X83 is I and X117is I; the residue corresponding to X117 is A and X122 is T; the residuecorresponding to X117 is E and X386 is D; the residue corresponding toX38 is S, X83 is I and X117 is L; the residue corresponding to X38 is S,X75 is L, X83 is I, X117 is I and X255 is P; the residue correspondingto X38 is S, X83 is I, X117 is I and X119 is V; the residuecorresponding to X38 is S, X40 is Y, X83 is I and X117 is A; the residuecorresponding to X38 is S, X83 is I, X117 is A and X251 is D; theresidue corresponding to X38 is S, X83 is I, X117 is A and X251 is A;the residue corresponding to X38 is S, X83 is I, X117 is I, X153 is E,X251 is W, X295 is T, X296 is F and X297 is Y; the residue correspondingto X38 is S, X83 is I, X117 is I, X251 is A, X295 is N, X296 is F andX297 is W; the residue corresponding to X38 is S, X83 is I, X117 is A,X251 is Y, X295 is T, X296 is F and X297 is W; the residue correspondingto X38 is S, X83 is I, X117 is I, X295 is T and X296 is A; the residuecorresponding to X38 is S, X83 is I, X117 is F and X251 is S; theresidue corresponding to X38 is S, X83 is I, X117 is L, X209 is D, X251is S, X376 is K and X400 is T; the residue corresponding to X38 is S,X83 is I, X117 is F, X251 is V and X376 is I; the residue correspondingto X38 is S, X83 is I, X117 is F, X251 is S, X295 is N, X296 is G andX297 is F; the residue corresponding to X28 is P, X83 is I, X117 is Aand X251 is V; the residue corresponding to X83 is I, X117 is N, X295 isT, X296 is G and X297 is F; the residue corresponding to X83 is I, X117is S, X251 is V, X296 is R and X297 is F; the residue corresponding toX38 is S, X83 is I, X117 is N and X251 is V; the residue correspondingto X38 is S, X83 is I, X117 is A and X330 is Y; the residuecorresponding to X38 is S, X40 is E, X75 is S, X83 is I and X117 is I;the residue corresponding to X38 is S, X83 is I and X117 is I; and theresidue corresponding to X83 is E, X117 is I and X333 is A.
 9. Themethod of claim 1, wherein said enone reductase polypeptide furthercomprises the following substitutions X38 is S, X83 is I, X117 is F,X251 is S, X295 is N, X296 is G and X297 is F; or the residuecorresponding to X38 is S, X83 is I, X117 is N and X251 is V.
 10. Themethod of claim 1, wherein said enone reductase polypeptide furthercomprises the following substitutions: X38 is S, X83 is I, X117 is F,X251 is S, X295 is N, X296 is G and X297 is F.
 11. The method of claim1, wherein said enone reductase polypeptide further comprises asubstitution or substitution set selected from: the residuecorresponding to X117 is A; the residue corresponding to X117 is I; theresidue corresponding to X117 is C; the residue corresponding to X117 isV the residue corresponding to X83 is I and X117 is I; the residuecorresponding to X117 is A and X122 is T; the residue corresponding toX38 is S and X117 is A; the residue corresponding to X38 is S, and X83is I, and X117 is I; the residue corresponding to X38 is S, and X83 isI, and X117 is L; the residue corresponding to X28 is P, X83 is I, X117is A, and X251 is V; the residue corresponding to X38 is S, X83 is I,X117 is A and X251 is D; the residue corresponding to X38 is S, X83 isI, X117 is A and X251 is A; the residue corresponding to X38 is S, X40is Y, and X83 is I and X117 is A; the residue corresponding to X38 is S,X83 is I, X117 is A and X330 is Y; the residue corresponding to X38 isS, X83 is I, X117 is I and X119 is V; the residue corresponding to X38is S, X40 is E, X75 is S, and X83 is I and X117 is I; the residuecorresponding to X38 is S, X83 is I, X117 is I, D295 is T and X296 is A;the residue corresponding to X38 is S, X75 is L, X83 is I, X117 is I andX255 is P; and the residue corresponding to X38 is S, X83 is I, X117 isI, X251 is A, X295 is N, X296 is F and X297 is W.
 12. The method ofclaim 1, wherein said enone reductase polypeptide further comprisessubstitution selected from: the residue corresponding to X117 is E andX386 is D; the residue corresponding to X83 is I, X117 is S, X251 is V,X296 is R and X297 is F; the residue corresponding to X38 is S, X83 isI, X117 is F and X251 is S; the residue corresponding to X38 is S, X83is I, X117 is F, X251 is S, X295 is N, X296 is G and X297 is F; theresidue corresponding to X38 is S, X83 is I, X117 is L, X209 is D, X251is S, X376 is K, and 400 is T; the residue corresponding to X38 is S,X83 is I, X117 is N and X251 is V; and the residue corresponding to X83is I, X117 is N, X295 is T, X296 is G and X297 is F.
 13. The method ofclaim 1, wherein said enone reductase polypeptide comprises a furthersubstitution selected from: the residue corresponding to X38 is S andX117 is A; the residue corresponding to X83 is I and X117 is I; theresidue corresponding to X117 is A and X122 is T; the residuecorresponding to X38 is S, and X83 is I and X117 is I; the residuecorresponding to X83 is E, X117 is I and X333 is A; the residuecorresponding to X38 is S, X83 is I, X117 is A and X251 is D; theresidue corresponding to X38 is S, X83 is I, X117 is A, X251 is A; theresidue corresponding to X38 is S, X75 is L, X83 is I, X117 is I andX255 is P; the residue corresponding to X38 is S, X83 is I, X117 is Iand X119 is V; the residue corresponding to X83 is I, X117 is S, X251 isV, X296 is R, X297 is F; the residue corresponding to X38 is S, X83 isI, X117 is A, and X330 is Y; the residue corresponding to X38 is S, X40is E, X75 is S, X83 is I and X117 is I; the residue corresponding to X83is I, X117 is N, X295 is T, X296 is G and X297 is F; and the residuecorresponding to X28 is P, X83 is I, X117 is A and X251 is V.
 14. Themethod of claim 13, wherein said enone reductase polypeptide furthercomprises substitutions at the residues corresponding to the following:X38 is S, X83 is I, X117 is F, X251 is S, X295 is N, X296 is G and X297is F.
 15. The method of claim 1, wherein said enone reductasepolypeptide further comprises a substitution set selected from: theresidue corresponding to X117 is E and X386 is D; the residuecorresponding to X28 is P, X83 is I, X117 is A and X251 is V; theresidue corresponding to X38 is S, X83 is I, X117 is A and X330 is Y;and the residue corresponding to X38 is S, X83 is I, X117 is F and X251is S.
 16. The method of claim 1, wherein said enone reductasepolypeptide further comprises a substitution set selected from: X38 isS, X83 is I, X117 is A, X251 is Y, X295 is T, X296 is F, X297 is W; andX38 is S, X83 is I, X117 is I, X251 is A, X295 is N, X296 is F, and X297is W.
 17. The method of claim 1, wherein said enone reductasepolypeptide further comprises a substitution at position W117.
 18. Themethod of claim 17, wherein said enone reductase polypeptide furthercomprises one of the following substitutions W117A/C/E/I/L/M/N/Q/V/F/X.19. The method of claim 1, wherein said enone reductase is an Old YellowEnzyme polypeptide.
 20. The method of claim 1, wherein said enonereductase is a chimeric Old Yellow Enzyme polypeptide.